Prognostic implications of PCSK9 expression in HER2-positive breast cancer

Introduction

Approximately 20 % of invasive breast cancers (BC) are human epidermal growth factor receptor 2 (HER2)-positive. These tumors are highly malignant and have demonstrated sensitivity to anti-HER-2 targeted therapy [1]. HER-2 positive is defined as immunohistochemistry (IHC) 3+ or IHC 2+ with HER-2 amplification and fluorescence in situ hybridization (FISH) of the HER-2 protein as the gene amplified phenotype. And neoadjuvant chemotherapy (NACT) plus neoadjuvant anti-HER-2 therapy is an effective therapy for locally advanced HER2-positive BC patients [2]. Pathological complete remission (pCR) achieved during surgery is frequently used as an endpoint in clinical trials and serves as a predictor of favorable prognosis in neoadjuvant HER2-positive patients [3]. The most widely accepted definition of pCR is the absence of invasive cancer in the breast and axillary lymph nodes, regardless of residual ductal carcinoma in situ (DCIS) (ypT0/is ypN0).

The intricate tumorigenesis process encompasses immune system-tumor interactions. Research on tumor metabolism has revealed that tumor cells undergo significant metabolic alterations compared to normal cells, primarily characterized by the “Warburg effect”. This phenomenon creates a hypoxic, acidic, and nutrient-deprived tumor microenvironment (TME), which in turn enhances lipid metabolism in tumor cells to satisfy their heightened energy demands [4]. Under hypoxic conditions, lipoproteins and their receptors are transiently upregulated, leading to the transfer of lipoproteins to tumor cells and an increase in lipid storage to reinforce the tumor-forming ability of tumor cells [5]. Meanwhile, the altered metabolic pattern of the tumor cells also exerts an influence on immune cell metabolism and function in the TME, which not only lowers immune cell proliferation and differentiation but also affects immune cell infiltration and reduction of effector function, further driving the malignant biological behavior of the tumor cells [6], 7]. Increasing evidence indicates that cholesterol plays a pivotal role in cancer development. Targeting molecules related to cholesterol metabolism can effectively inhibit tumor growth, remodel TME, and restore anti-tumor immunity [8], 9]. BC is no exception, with some studies describing a relationship between low-density lipoprotein (LDL) cholesterol (LDL-C) and BC and others showing no association [10], [11], [12], [13].

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is critical in modulating cholesterol metabolism homeostasis. It facilitates LDL receptor (LDLR) degradation, improving lipid levels [14]. PCSK9 antibodies (evolocumab and alirocumab) are effective for patients with hypercholesterolemia. Growing studies have indicated PCSK9 overexpression in various tumors; it promotes tumor cell proliferation and inhibits apoptosis, facilitating tumor initiation and progression, contributing to tumor recurrence and treatment resistance [15], 16]. Beyond that, PCSK9 expression in tumors has been shown to decrease significant major histocompatibility protein class I (MHC-I) expression or mediate T cell receptor (TCR) degradation. Silencing PCSK9 expression facilitates intra-tumor infiltration and effector function of cytotoxic T cells, enhancing immunotherapy’s anti-tumor effect [17], 18]. PCSK9 can also suppress anti-tumor immunity by influencing lactate metabolism in cancer cells and driving tumor-associated macrophage (TAM) polarization toward the M2 phenotype [19]. Nevertheless, little is known about PCSK9 expression and its underlying therapeutic value in HER2-positive BC. More importantly, the lipid metabolism-associated gene fatty acid synthase (FASN) is overexpressed in HER2-positive BC, which is rare in other BC subtypes, linking to bad prognosis of HER2-positive patients [20], 21]. In a mouse model, PCSK9 was found to promote tumor development by influencing the expression of FASN, thereby inhibiting cancer cell apoptosis via the Bax/Bcl-2/caspase-9/caspase-3 pathway [22]. Thus, it is essential to investigate this approved lipid-lowering target’s role and potential therapeutic value in HER2-positive BC.

Hereby, the current study focuses on the key genes of lipid metabolism in the TME, analyzes PCSK9’s role in HER-2-positive BC, and detects PCSK9 protein expression before and after neoadjuvant therapy through bioinformatics, analyses its relationship with clinicopathological features and prognosis, and understands the influence of the expression and changes of PCSK9 on the prognosis of HER2-positive BC to lay a theoretical foundation for the search of targeted lipid metabolism therapy or adjuvant immunotherapy for HER2-positive BC.

Materials and Methods

Results

PCSK9 expression’s impact on the survival of BC patients

The variance in PCSK9 expression within BC may influence patients’ prognoses, as shown in Figure 1A. The PCSK9 high expression group showed higher survival rate compared with the low expression group. Nevertheless, there was no significant difference (p>0.05). Although there was also no significant correlation between PCSK9 expression and the survival rate of each molecular subtype of BC (p>0.05), the PCSK9 low expression group exhibited higher 5-year survival rate relative to the PCSK9 high expression group in HER2-positive patients (Figure 1B). These findings implied that the distinct expression patterns of PCSK9 might affect patients’ prognosis across different BC subtypes.

Figure 1:

Correlation between PCSK9 levels and patient survival. (A) Effect of PCSK9 levels on survival of BC patients. (B) Correlation between PCSK9 levels and survival in different BC subtypes.

Correlation of the expression of PCSK9 with clinicopathological variables

The Wilcoxon rank-sum test revealed a significant elevation in PCSK9 expression in tumor samples relative to normal samples (Figure 2A). Consistently, similar findings were obtained in paired analyses of the patient’s normal and tumor tissues (Figure 2B). Furthermore, the association between PCSK9 and FASN expression and the molecular subtypes of tumors was further assessed. According to the results, PCSK9 expression levels, as well as the expression levels of TNBC FASN, were notably different in patients with luminal B type compared to the other three types (all p-values <0.001) (Figure 2C and D). And there was no remarkable correlation between PCSK9 expression and clinicopathological features of HER2-positive patients (Figure 2E–H). The relationship between FASN and PCSK9 was also analyzed and no significant linear relationship was found (Figure 2I).

Figure 2:

Differential PCSK9 expression in samples and its correlation with survival and clinicopathological staging characteristics of HER2-positive patients. (A) Differential PCSK9 expression in normal and tumor samples. All samples were analyzed via Wilcoxon rank-sum test. (B) Paired analysis of PCSK9 expression in normal and tumor samples from the same patient (by Wilcoxon rank sum test). (C, D) PCSK9 and FASN expression in different molecular subtypes of BC were analyzed via Kruskal–Wallis rank-sum test. (E–H) The correlation of PCSK9 expression with clinicopathological staging of HER2-positive patients was tested for significance based on the Wilcoxon rank-sum test or Kruskal–Wallis rank-sum test. (I) Linear association of PCSK9 with FASN in HER2-positive BC, correlation test by Spearman coefficient. Significance levels are indicated by asterisks: ***p<0.001.

PCSK9 may be a biomarker of the regulation of the TME in HER2-positive BC

Given the negative correlation between PCSK9 levels and survival in HER2-positive patients, GSEA was conducted for both groups, relative to the median PCSK9 expression. As shown in Figure 3A, in the Hallmark gene set, genes in the low expression group were primarily enriched in pathways associated with immune (isograft rejection, interferon response, etc.). However, genes in another group were enriched in the coagulation pathway (Figure 3B). Several gene sets about immune function were reinforced in the low expression group for the C7 set (i.e., the immune gene set) (Figure 3C). However, the high-expression group enriched only a few gene sets (Figure 3D). These results implied that PCSK9 may potentially indicate TME condition in HER2-positive patients.

Figure 3:

GSEA of PCSK9 expression samples. (A) GSEA on PCSK9 low expression samples collected at Hallmark. Each line with a specific color indicates a gene set, with upregulated genes on the coordinates origin’s left side and those downregulated on the x-axis’ right side. NOM p<0.05 and FDR q<0.06 indicated a significant gene set. (B) GSEA on PCSK9 high expression sample Hallmark. (C) Samples from the PCSK9 low expression group showed a high enrichment in C7 (i.e. the immune gene set). Only a few leading gene sets are shown in the graph. (D) Samples from the PCSK9 high expression group showed less enrichment in C7 for the gene set.

Correlation between the PCSK9 and the TIC ratio

To substantiate the PCSK9 expression-immune microenvironment correlation, TIC subpopulations’ proportion was assessed by utilizing the CIBERSORT algorithm. This analysis generated 21 TIC profiles in HER2-positive tumor specimens (Figure 4A and B). Difference and correlation analyses indicated that only γδ T cells were significantly related to PCSK9 expression (p<0.05) (Figure 4C). Taken together, PCSK9 might exert an influence on the immune activity of TME in HER2-positive patients by affecting the proportion of γδ T cells.

Figure 4:

Correlation of TIC ratio with PCSK9 expression. (A) Bar graph showing 21 TICs in HER2-positive tumor specimens. The graphs’ column names include sample IDs. (B) Violin plots show the proportional differentiation of 21 immune cells between low and high PCSK9-expressing HER2-positive tumor samples compared with the median PCSK9 expression, with the significance tested through Wilcoxon rank sum. (C) Scatter plots manifest a correlation between only 1 TIC ratio and PCSK9 expression (p<0.05). A linear model is fitted to the red line in each plot to show the convergence of the ratio of immune cells to PCSK9 expression and correlation tests were performed using the Spearman coefficient. Significance levels are indicated by asterisks: ns, no significance; **p<0.01.

PCSK9 expression before neoadjuvant therapy correlates with clinicopathological features

The rate of positive PCSK9 expression in pre-neoadjuvant HER2-positive BC specimens was 100.0 %. Statistical analysis indicated that pre-neoadjuvant PCSK9 expression (pre-PCSK9) was positively correlated with histological grade (p=0.007) and HR status (p=0.017) (Table 3).

Table 3:

Comparison of baseline characteristics between patients in the group with low pre-PCSK9 expression and the group with high pre-PCSK9 expression n (%).

Variables		n=92	Low (n=64)	High (n=28)	p-Value
Age, years	<50	43 (46.7 %)	28 (43.8 %)	15 (53.6 %)	0.385
	≥50	49 (53.3 %)	36 (56.2 %)	13 (46.4 %)
Tumor size, cm	≤5	57 (62.0 %)	39 (60.9 %)	18 (64.3 %)	0.761
	>5	35 (38.0 %)	25 (39.1 %)	10 (35.7 %)
TNM stage	II	48 (52.2 %)	35 (54.7 %)	13 (46.4 %)	0.466
	III	44 (47.8 %)	29 (45.3 %)	15 (53.6 %)
Grade	I/II	78 (84.8 %)	59 (92.2 %)	19 (67.9 %)	0.007
	III	14 (14.2 %)	5 (7.8 %)	9 (32.1 %)
HR	–	33 (35.9 %)	28 (43.8 %)	5 (17.9 %)	0.017
	+	59 (64.1 %)	36 (56.2 %)	23 (82.1 %)
Ki-67	<20 %	3 (3.3 %)	1 (1.6 %)	2 (7.1 %)	0.168
	≥20 %	89 (96.7 %)	63 (98.4 %)	26 (92.9 %)
HER2	2+	14 (15.2 %)	7 (10.9 %)	7 (25.0 %)	0.084
	3+	78 (84.8 %)	57 (89.1 %)	21 (75.0 %)

Survival analysis between different subgroups of HER2-positive patients treated with neoadjuvant therapy (Figure 5A).

Figure 5:

Intensity of IHC staining for PCSK9 and its correlation with survival time in HER2-positive patients. (A) From left to right, the stains are light brown, brown-yellow, and tan in this order. The numbers in the lower left corner represent the magnifications. (B)The OS of the PCSK9 high and low expression groups of HER2-positive patients was subjected to K–M survival analysis. p=0.44 by log-rank test. (C)The K-M method (log-rank test p=0.765) was used to analyze DFS in HER2-positive patients.

The 92 cases of HER2-positive patients in Zhongshan City People’s Hospital had detailed records of follow-up time and were grouped by high and low PCSK9 expression for OS (Figure 5B) and DFS (Figure 5C) survival analyses, among which three cases were lost to follow-up; the remaining 89 cases were followed up from 6 to 82 months (median follow-up time of 28 months), and 15 cases have relapsed, among which seven cases died.

Discussion

The TME is pivotal in the initiation and progression of tumorigenesis. In vivo, lipid accumulation can occur within the TME, and cholesterol, crucial for cell proliferation, can contribute to the formation of an immunosuppressive microenvironment. Interestingly, PCSK9 may have a strong appeal as an oncogenic factor due to its crucial effects on regulating cholesterol homeostasis and anti-tumor immunity. Hereby, low PCSK9 expression was linked with BC patients’ poor prognosis, starting from transcriptomic analyses in the UALCAN-TCGA database. Nevertheless, the outcomes varied among patients with HER2-positive molecular subtype. Consequently, we placed additional emphasis on investigating PCSK9’s role in HER2-positive BC. A pan-cancer analysis of PCSK9 showed that PCSK9 was highly expressed in several tumor types and it should be considered an important prognostic factor for the [24]. In this study, PCSK9 expression levels were also noticeably elevated in BC tissues relative to normal tissues, and PCSK9 and FASN levels were significantly correlated with the molecular subtype of BC (p<0.05): The reason for the lowest level of PCSK9 expression in luminal B patients, which significantly differed from TNBC and HER2-positive patients, might be related to the relatively low malignancy of luminal B BC; The lowest level of FASN expression was found in TNBC. Similar results have also been evidenced previously [25]. However, its expression level was not the highest in HER2-positive patients, and the highest level was found in luminal B. The hypothesis posited that the expression levels of FASN in the luminal B subtype could potentially be suppressed during translation. Studies of HCC reported that PCSK9 might affect the expression of FASN [22], whereas, in this study, no significant linear relationship was observed between PCSK9 and FASN (p>0.05), which might be attributed to the relationship between the two may vary in different tumors.

According to a study published in the scientific journal Nature, PCSK9 facilities MHC-I degradation by lysosome mediation in cancer cells, inhibiting MHC-I recycling back to the membrane, resulting in evading cytotoxic T cell (CTL) surveillance, and ultimately reducing lymphocyte infiltration and tumor immune evasion [17]. Furthermore, LDLR could increase cell surface levels through direct interaction with the TCR complex [18]. Tumor cell-derived PCSK9 in the TME attenuates lymphocyte effector function by binding to LDLR and inhibiting LDLR and TCR recirculation to plasma membrane. This process downregulates T cell LDLR levels and TCR signaling. PCSK9 inhibition attenuates tumor growth in an LDLR-dependent manner. To sum up, PCSK9 could independently or synergistically control tumor growth via the MHC-I and LDLR pathways. While γδ T cells are excellent anticancer effectors and may target tumors broadly, they can sense conserved cellular stress signals that are common in transformed cells, with Vγ9Vδ2 T cells being the most abundant subpopulation of human γδ T cells. It has been reported that monitoring γδ T cells could detect transformed cells showing a combination of mevalonate pathway hyperactivity and reduced oxidative phosphorylation activity. The study elucidated the mechanism by which cellular stress can facilitate cancer cells-Vγ9Vδ2 T cell interactions [26]. As indicated by a series of bioinformatic analysies results in this study, PCSK9 may indicate TME condition in HER2-positive patients and that it might influence the immunoreactivity of TME in HER2-positive patients by affecting the ratio of γδ T cells, which could be explained by the fact that the oncogenic effect of PCSK9 is related to the accumulation of cholesterol in the tumor and that lowering PCSK9 induces cancer cells to synthesize lipids to meet their own needs via the mevalonate pathway, leading to a corresponding increase in the proportion of γδ T cells. As a result, PCSK9 is a possible biomarker for patient’s prognosis, as well as treatment target for TME in HER2-positive patients. However, this study did not investigate the detailed mechanism by which PCSK9 inhibits the regulation of the immune cell population γδ T cells.

In recent years, although neoadjuvant therapy has achieved significant progress with the advent and use of dual-targeted drugs, the prognosis for patients who do not achieve pathological pCR after treatment remains poor. Therefore, it is crucial to explore novel treatment biomarkers and investigate their roles in tumorigenesis to enhance patient survival. In a Mendelian randomized study of BC, the relationship between LDL-C levels and disease risk was evaluated by modeling that inhibition of PCSK9 lowers LDL-C and thus reduces BC risk [27]. Furthermore, low PCSK9 expression has been linked to favorable prognosis and have been reported to upregulate with the severity of breast disease [28]. Nevertheless, little is known about the relationship between efficacy and PCSK9 expression in intermediate and advanced HER2-positive BC tumor tissue. Building upon this background, this research retrospectively reviewed clinical data from a cohort of patients with intermediate to advanced HER2-positive BC who underwent neoadjuvant therapy. We examined PCSK9 expression in pre-treatment tumor tissue by virtue of IHC to assess whether PCSK9 expression in HER2-positive tumor tissue correlated with treatment response. Our study showed that PCSK9 expression was an influential but not an independent predictor of achieving pCR after neoadjuvant therapy (p=0.05), and it was considered that this might be related to the small sample size. Further analysis of PCSK9 expression using clinicopathological data revealed a positive correlation between PCSK9 and tumor histological stage and HR status [19], 29]. In the end, we observed that highly expressed PCSK9 was linked to undesirable prognosis in HER2-positive patients, which conformed to transcriptomic findings, especially in the small dataset of HER2-positive patients. Nevertheless, the potential of PCSK9 as a biomarker ought to be validated by further studies and clinical trials, and its potential to guide immunotherapeutic interventions in tumor therapy should be explored.

However, there exist some limitations, firstly, the sample size of both the downloaded transcriptome sequencing data and the data from the clinical study in our center is small, secondly, the neoadjuvant treatment regimen, insufficient patient follow-up, failure to detect TIL levels in tumor tissues, and lack of artificial intelligence correction for the interpretation of experimental results.

Conclusions

In conclusion, our study indicated that PCSK9 promised to be a marker for TME condition in HER2-positive patients. This, in turn, might impact the immunological activity of the TME in HER2-positive patients by influencing the ratio of γδ T cells. Moreover, highly expressed PCSK9 in cancerous tissue is a detrimental reason and a poor prognostic marker for neoadjuvant therapy in patients with intermediate and advanced HER2-positive BC.