Physical and functional interactions between LDLR family members and CXCR4 in breast cancer

Jiankang Zhang; Jinxiao Chen; Da Wo; En Ma; Hongwei Yan; Jun Peng; Dan‐ni Ren; Weidong Zhu

doi:10.1111/febs.70016

. 2025 Feb 28;292(17):4580–4590. doi: 10.1111/febs.70016

Physical and functional interactions between LDLR family members and CXCR4 in breast cancer

Jiankang Zhang ^1,², Jinxiao Chen ¹, Da Wo ¹, En Ma ¹, Hongwei Yan ³, Jun Peng ¹, Dan‐ni Ren ^1,^✉, Weidong Zhu ^1,^✉

PMCID: PMC12414868 PMID: 40022442

Abstract

C‐X‐C chemokine receptor type 4 (CXCR4) belongs to the seven‐span G protein‐coupled receptor family and plays an important role in promoting cancer metastasis. The single‐span receptor, low‐density lipoprotein receptor‐related protein 6 (LRP6) is commonly considered to be a co‐receptor of Wnt and plays an indispensable role during animal development. We recently demonstrated that LRP6 directly binds to CXCR4 via its ectodomain and prevents CXCR4‐induced breast cancer metastasis. As a result of structural similarity, LRP6 is also categorized within the low‐density lipoprotein receptor (LDLR) family that is involved in lipoprotein transport. We therefore explored whether other LDLR family members could interact with CXCR4. Immunoprecipitation and western blotting analysis showed that LDLR and very low‐density lipoprotein receptor (VLDLR) physically interacted with CXCR4. Although stromal cell‐derived factor 1/CXCR4 signaling was inhibited by LDLR and LRP1, it was promoted by VLDLR, LRP8 and apolipoprotein E, a general agonist of the LDLR family. Furthermore, breast cancer patients with high CXCR4 expression exhibited the worst prognosis only when combined with high levels of VLDLR/LRP8/apolipoprotein E or low expression of LDLR/LRP1, further suggesting distinct positive and negative roles of LDLR family members in regulating CXCR4. Additional members of the LDLR family, SORL1 and LRP2, also showed a negative correlation with CXCR4 in the prognosis of breast cancer patients. The findings of the present study show that the LDLR family can regulate CXCR4, endowing its members with a previously undescribed role, also suggesting their potential as new breast cancer therapeutic targets and prognostic markers.

Keywords: breast cancer, CXCR4, LDLR family, prognosis, SDF‐1

Here, we demonstrate that members of the low‐density lipoprotein receptor (LDLR) family can regulate C‐X‐C chemokine receptor type 4 (CXCR4) signaling and therefore affect CXCR4‐induced breast cancer metastasis. We found that very low‐density lipoprotein receptor (VLDLR), low‐density lipoprotein receptor‐related protein 8 (LRP8) and apolipoprotein E (ApoE) could activate the stromal cell‐derived factor 1 (SDF‐1)/CXCR4 signaling pathway and promote cancer cell metastasis. Conversely, other members of the LDLR family such as LDLR, LRP6, LRP1 and SORL1 could inhibit SDF‐1/CXCR4 signaling and prevent cancer cell metastasis. Our findings suggest that the LDLR family could have potential as new breast cancer therapeutic targets and prognostic markers.

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Abbreviations

ApoE: apolipoprotein E
CM: conditioned medium
Co‐IP/WB: co‐immunoprecipitation/western blot
CXCR4: C‐X‐C chemokine receptor type 4
ERK1/2: extracellular signal‐regulated kinase 1/2
GPCR: G protein‐coupled receptor
LDLR: low‐density lipoprotein receptor
LRP6: LDL receptor‐related protein 6
SDF‐1: stromal cell‐derived factor 1
TNBC: triple‐negative breast cancer
VLDLR: very low‐density lipoprotein receptor

Introduction

C‐X‐C chemokine receptor 4 (CXCR4) is a G protein‐coupled receptor (GPCR) that is involved in multiple physiological and pathological processes upon binding to its ligand, stromal cell‐derived factor 1 (SDF‐1). CXCR4 is upregulated in multiple tumors, such as breast cancer, prostate cancer, melanoma and colorectal cancer. SDF‐1/CXCR4 plays pivotal roles in cancer progression via promoting tumor growth, metastasis, angiogenesis or therapeutic resistance [1, 2]. The SDF‐1/CXCR4 signaling pathway significantly correlates with survival of different type of cancer patients [3, 4] and its inhibition prevents tumor progression [5, 6]. As a result, multiple CXCR4‐targeted drugs have been developed, such as FDA‐approved Pplerixafor for non‐Hodgkin's lymphoma and multiple myeloma, as well as BKT140 and BMS‐936564, which are under clinical trial for multiple myeloma, acute myelogenous leukemia and selected B‐cell cancers. The SDF‐1/CXCR4 signaling pathway also plays important roles in physiological status, such as the development of hematopoietic cells and early human T‐cells [7]. Homozygous CXCR4 knockout mice indeed die perinatally [8].

We recently demonstrated that low‐density lipoprotein receptor‐related protein 6 (LRP6) via its ectodomain directly binds to CXCR4 and inhibit SDF‐1/CXCR4 signaling pathway [9]. LRP6 is commonly regarded as a Wnt co‐receptor and key activator of Wnt/β‐catenin signaling pathway [10]. As a result of structural similarity, LRP6 is also categorized to the single‐span low‐density lipoprotein receptor (LDLR) family [11], which includes 13 members, such as low‐density lipoprotein receptor (LDLR), very low‐density lipoprotein receptor (VLDLR), LRP1 and LRP8. The major structural similarity of LDLR family members lies in their extracellular domains, containing LDLR type A repeats and epidermal growth factor‐like domain (Fig. 1A). For example, the extracellular domain of LRP6 is composed of four LDLR type A repeats and three epidermal growth factor‐like domains [12]. As a result of their structural similarity, we hypothesized that, similar to LRP6, other LDLR family members were also able to physically and functionally interact with CXCR4. We investigated the interaction between several LDLR family members and CXCR4, and found that multiple LDLR family members had an ability to affect SDF‐1/CXCR4 signaling pathway.

Fig. 1 — Interaction between LDLR/VLDLR and CXCR4. (A) Schematic diagram of LDLR, VLDLR, LRP6 and LRP6‐ΔN. (B) HEK293 cells were transfected with Flag‐CXCR4 and either Myc‐VLDLR, Myc‐LDLR or Myc‐LRP6‐ΔN. Forty‐eight hours post‐transfection, co‐immunoprecipitation (Co‐IP) and western blot (WB) analyses were performed using Flag or Myc antibodies. Myc‐LRP6‐ΔN served as a negative control. Specific bands for myc‐VLDLR/myc‐LDLR/myc‐LRP6‐ΔN are indicated with a black arrow. The results are representative of three independent experiments. (C) WB analysis of SDF‐1‐induced signal transduction in Vector (pcDNA3.1), LRP6, VLDLR or LDLR‐overexpressed HepG2 cells after 48 h of post‐transfection. Cells were serum‐starved in 0.5% fetal bovine serum for 24 h and SDF‐1 was treated for 30 min. The results are representative of three independent experiments. (D) Photo images and quantification of transwell analysis of cell migration following transfection of CXCR4 with or without LDLR in 4T1 cells. Data are presented as mean ± standard deviation (SD). *P < 0.05, one‐way analysis of variance. Scale bar = 50 μm (n = 3). (E) WB analysis of Flag‐CXCR4 distribution in the absence or presence of myc‐VLDLR in Cos7 cells at 48‐h post‐transfection. Vectors (pEnter for CXCR4 and pcDNA3.1 for VLDLR) were cotransfected to ensure equal amounts of plasmid DNA in each sample. Numbers indicate densitometry analysis. C, cytoplasmic fraction; M, membrane fraction; T, total protein. The results are representative of three independent experiments. (F) Immunofluorescence analysis of the localization of GFP‐CXCR4 after transfection with or without Myc‐VLDLR for 48 h in HEK293 cells. The results are representative of three independent experiments. (G) WB analysis of Flag‐CXCR4 distribution in the absence or presence of Myc‐LDLR in Cos7 cells. Numbers indicate densitometry analysis. C, cytoplasmic fraction; M, membrane fraction; T, total protein. The results are representative of three independent experiments. (H, I) Kaplan–Meier analysis of overall survival in breast cancer patients (dataset: NKI295). Patients were divided according to the expression of LDLR (H) or LDLR and CXCR4 (I), as indicated. (J, K) Kaplan–Meier analysis of overall survival in breast cancer patients (dataset: NKI295). Patients were divided according to the expression of VLDLR (J) or VLDLR and CXCR4 (K), as indicated.

Results

LDLR and VLDLR interact with CXCR4 and influence the SDF‐1/CXCR4 signaling pathway

LDLR and VLDLR are typical members of LDLR family with the shortest ectodomains (Fig. 1A), which should be ideal for investigating the interaction between the LDLR family and CXCR4. We transfected Myc‐tagged LDLR or VLDLR plasmid together with Flag‐tagged CXCR4 plasmid into HEK293 cells and examined their interactions using co‐immunoprecipitation/western blot (co‐IP/WB assays). We found that both Myc‐VLDLR and Myc‐LDLR interacted with Flag‐CXCR4 (Fig. 1B). Additionally, endogenous LDLR and CXCR4 were co‐localized in murine 4T1 tumor (Fig. S1). We previously showed that, although LRP6 ectodomain was able to bind to a separate GPCR Frizzled, LRP6 cytoplasmic domain was not [13]. The co‐IP/WB assay showed that Myc‐LRP6‐ΔN did not bind to Flag‐CXCR4 either (Fig. 1B). Thus, the finding of Myc‐LRP6‐ΔN as a negative control validated the co‐IP/WB assay to determine the binding of Myc‐VLDLR or Myc‐LDLR to Flag‐CXCR4.

We further examined the functional interaction between these LDL receptors and CXCR4. Transfection of Myc‐LDLR as well as Myc‐LRP6 significantly inhibited SDF‐1‐induced c‐Jun and Akt phosphorylation in HepG2 cells (Fig. 1C). Overexpression of LDLR also inhibited SDF‐1‐induced cell migration in mouse breast cancer 4T1 cells (Fig. 1D) and human breast cancer MDA‐MB‐231 cells (Fig. S2). These results indicate that, similar to LRP6 [9], LDLR inhibits the SDF‐1/CXCR4 signaling pathway. Interestingly, VLDLR overexpression itself conversely activated c‐Jun and Akt, and further enhanced SDF‐1‐induced activations of c‐Jun and Akt (Fig. 1C), suggesting that, in contrast to LRP6 and LDLR, VLDLR has an ability to promote SDF‐1/CXCR4 signal transduction.

We further found that overexpression of Myc‐VLDLR for 48 h significantly downregulated membrane Flag‐CXCR4 expression and upregulated cytoplasmic Flag‐CXCR4 expression (Fig. 1E), suggesting that VLDLR has an ability to induce CXCR4 endocytosis. Furthermore, an immunofluorescence assay showed that GFP‐CXCR4 was mainly localized in the cell membrane when transfected alone (Fig. 1F). However, when co‐transfected with Myc‐VLDLR plasmid, GFP‐CXCR4 and Myc‐VLDLR were mainly expressed in the cytosol (Fig. 1F). These results indicate that VLDLR and CXCR4 can mutually regulate their locations from cell surface to cytoplasm. Of note, co‐transfection of Myc‐LDLR did not alter the localization of Flag‐CXCR4 (Fig. 1G). Additionally, LRP6 overexpression also unaltered the localization of Flag‐CXCR4 (data not shown). Thus, cytosolic translocation of CXCR4 induced by VLDLR may be required for VLDLR‐induced CXCR4 activation, although the precise underlying mechanism requires further investigation.

We further investigated the correlations of LDLR and VLDLR expression in the prognosis of breast cancer patients. We previously showed that high CXCR4 expression correlated with worsened prognosis for overall survival in the NKI295 dataset [14], which was further exacerbated in patients with low LRP6 expression but improved in patients with high LRP6 expression [9]. In the same dataset, we found that low LDLR expression showed a trend of worsened prognosis (P = 0.1376) (Fig. 1H). Interestingly, high CXCR4 expression correlated with worsened prognosis only in patients with low LDLR expression (P = 0.0254) but not in patients with high LDLR expression (P = 0.3723) (Fig. 1I). On the other hand, high VLDLR expression significantly correlated with worsened prognosis for overall survival in this dataset (P = 0.0302) (Fig. 1J). Intriguingly, high VLDLR expression only correlated with worsened prognosis in patients with high CXCR4 expression (P = 0.0050) but not in patients with low CXCR4 expression (P = 0.3123) (Fig. 1K). Furthermore, high CXCR4 expression only correlated with worsened prognosis in patients with high VLDLR expression (P = 0.0106) but not in patients with low VLDLR expression (P = 0.6075) (Fig. 1K). These results support our in vitro experimental findings, indicating that LDLR inhibits but VLDLR promotes the SDF‐1/CXCR4 signaling pathway.

LRP1 and LRP8 modulate the SDF‐1/CXCR4 signaling pathway

We also analyzed the relationship between LRP1 and CXCR4. Because LRP1 is a large protein containing 4544 amino acids [15], which is much larger than LRP6 (1613 amino acids) and leads to difficulty with respect to transfecting LRP1 plasmid, we observed its function by small interfering RNA knockdown instead of plasmid transfection. Knockdown of LRP1 enhanced SDF‐1‐induced activations of c‐Jun, extracellular signal‐regulated kinase 1/2 (ERK1/2) and p38 (Fig. 2A). Knockdown of LRP1 also significantly enhanced cell migration of 4T1 cells, which was inhibited by AMD3100, a CXCR4 antagonist (Fig. 2B). These results suggest that LRP1 inhibits the SDF‐1/CXCR4 signaling pathway.

By contrast to LRP1, knockdown of LRP8 prevented SDF‐1‐induced activations of c‐Jun, ERK1/2, Akt and p38 (Fig. 2A) and also inhibited cell migration in 4T1 cells (Fig. 2C). Intriguingly, co‐transfection of Flag‐LRP8 increased total as well as membrane protein levels of Flag‐CXCR4 (Fig. 2D), suggesting that LRP8 has the ability to stabilize CXCR4 protein. Supporting this notion, knockdown of LRP8 did not affect CXCR4 mRNA expression level (Fig. 2E). These results indicate that LRP8 is able to enhance the SDF‐1/CXCR4 signaling pathway by stabilizing CXCR4 protein.

We also examined the expression of LRP1 and LRP8 in the NKI295 dataset. Low expression of LRP1 significantly correlated with worsened prognosis for overall survival in breast cancer patients (P = 0.0331) (Fig. 2F). Of note, low LRP1 expression correlated with worsened prognosis only in patients with high CXCR4 expression (P = 0.0368) but not in patients with low CXCR4 expression (P = 0.9807) (Fig. 2G). Similalrly, high CXCR4 expression only correlated with worsened prognosis in patients with low LRP1 expression (P = 0.0325) but not in patients with high LRP1 expression (P = 0.5096) (Fig. 2G). On the other hand, patients with high LRP8 expression showed worsened prognosis for overall survival (P = 0.0050) (Fig. 2H). However, high LRP8 expression only correlated with worsened prognosis in patients with high CXCR4 expression (P = 0.0040) but not in patients with low CXCR4 expression (P = 0.7711) (Fig. 2I), whereas high CXCR4 expression only correlated with worsened prognosis in patients with high LRP8 expression (P = 0.0121) but not in patients with low LRP8 expression (P = 0.5862) (Fig. 2I). These results further support the inhibitory role of LRP1 and the promotional role of LRP8 in regulating the SDF‐1/CXCR4 signaling pathway.

Impact of other LDLR family members on SDF‐1/CXCR4 prognosis in breast cancer patients

We further analyzed the expression of other LDLR family members in the NKI295 dataset. Breast cancer patients with low expression of SORL1, also showed worsened prognosis (P = 0.0014) (Fig. 3A). However, low SORL1 expression correlated with worsened prognosis only in patients with high CXCR4 expression (P = 0.0024) but not in patients with low CXCR4 expression (P = 0.1072) (Fig. 3B). Similarly, high expression of CXCR4 correlated with worsened prognosis only in patients with low SORL1 expression (P = 0.0230) but not in patients with high SORL1 expression (P = 0.1801) (Fig. 3B). These results suggest an inhibitory role of SORL1 in regulating the SDF‐1/CXCR4 signaling pathway.

Low expression of LRP2 correlated with worsened prognosis in breast cancer patients (P = 0.0014) (Fig. 3C), suggesting a negative correlation of LRP2 expression in the prognosis of breast cancer patients. It is noteworthy to emphasize that high expression of CXCR4 correlated with worsened prognosis in breast cancer patients [9]. These results suggest an opposing role of LRP2 and CXCR4 in regulating the prognosis of breast cancer patients. Of note, the expression levels of CXCR4 became uncorrelated in the prognosis in patients with low LRP2 (Fig. 3D, red line and purple line). These results suggest a strong effect of LRP2 in regulating CXCR4. Importantly, the prognosis of even 100% survival in low CXCR4/high LRP2 (Fig. 3D, black line) suggests a highly promising therapy for complete prevention of breast cancer, which warrants further investigation.

Although high LRP5 expression or low LRP1B expression showed worsened prognosis, they were not correlated with CXCR4 expression in the prognosis of breast cancer patients (Fig. 3E–H). These results suggest that LRP5 accelerates tumor death and LRP1B prolongs survival of breast cancer patients in a CXCR4‐independent manner. In addition, expression of LRP4 was not related to overall survival or CXCR4‐induced tumor death (Fig. 3I,J).

Apolipoprotein E (ApoE) as a ligand of the LDLR family influences the SDF‐1/CXCR4 signaling pathway

We next investigated the role of ApoE that is a general ligand of the LDLR family members [16]. Overexpression of ApoE enhanced SDF‐1‐induced activations of c‐Jun, ERK1/2 and Akt (Fig. 4A). Similarly, pretreatment with ApoE‐conditioned medium (CM) but not Vector‐CM also enhanced SDF‐1‐induced activations of c‐Jun, ERK1/2 and Akt in both HepG2 cells (Fig. 4B) and 4T1 cells (Fig. 4C). These results suggest that ApoE has the ability to promote SDF‐1/CXCR4 signal transduction.

Fig. 4 — Cross‐talk between ApoE and the SDF‐1/CXCR4 signaling pathway. (A) HepG2 cells were transfected with ApoE or Vector for 24 h, serum‐starved in 0.5% fetal bovine serum‐containing Dulbecco's modified Eagle's medium for 24 h, and then treated with SDF‐1. Western blot analysis was performed to assess SDF‐1‐induced signaling. n = 3. (B, C) WB analysis of SDF‐1‐induced signal transduction with or without pretreatment of ApoE‐conditioned medium (CM) in HepG2 cells (B) or 4T1 cells (C). Vector‐CM and ApoE‐CM were serum‐free medium collected from Vector or ApoE transfected HEK293 cells at 72 h post‐transfection. HepG2 or 4T1 Cells were pretreated with either Vector‐CM or ApoE‐CM for 4 h before SDF‐1 treatment. n = 3 for (B) and (C). (D, E) Kaplan–Meier analysis of overall survival in breast cancer patients (dataset: NKI295). Patients were divided according to the expression of ApoE (D) or ApoE and CXCR4 (E), as indicated.

We further analyzed the role of ApoE in the prognosis of breast cancer patients in the NKI295 dataset. Patients with high ApoE expression showed worsened prognosis for overall survival (P = 0.00085) (Fig. 4D). Similarly to LRP8 and VLDLR, high ApoE expression only correlated with worsened prognosis in patients with high CXCR4 expression (P = 0.0198) but not in patients with low CXCR4 expression (P = 0.7381) (Fig. 4E). Furthermore, the prognosis of patients with high CXCR4 expression was worsened in high ApoE expression (P = 0.0475) but not in low ApoE expression (P = 0.4776) (Fig. 4E). These results further indicate the promotional role of ApoE in regulating SDF‐1/CXCR4 signaling pathway. Of note, ApoE has been reported to be able to induce downregulation of membrane LRP8 but not LRP1 and LDLR [17]. Therefore, the ability of ApoE in enhancing SDF‐1/CXCR4 signal transduction may be via binding to its receptors such as LRP1 and LDLR and by relieving their inhibitory effects on the SDF‐1/CXCR4 signaling pathway. We further conducted protein–protein interaction analyses between CXCR4 and LDLR family members via molecular docking models as predicted by AlphaFold 3 [18] (Fig. S3A–J). Most predicted template modeling scores were 0.5 or higher, suggesting potentially accurate prediction of interactions. These computational predictions, in combination with our experimental data, strongly support the conclusion that CXCR4 interacts with LDLR family members.

Discussion

In the present study, we revealed an unexpected role of the single‐span lipoprotein receptor family that physically and functionally interacted with seven‐span GPCR CXCR4. Importantly, multiple LDLR family members via regulating CXCR4 showed a highly significant correlation in the prognosis of breast cancer patients. Although, similar to its name, LDLR family is generally considered to account for lipoprotein transfer, our current findings might open a new avenue for the study of the LDLR family.

Interestingly, the LDLR family members regulated CXCR4 with different mechanisms and gave rise to completely opposing effect on CXCR4‐induced breast cancer progression. LDLR, LRP1 and SORL1 inhibited but VLDLR, LRP8 and ApoE promoted the SDF‐1/CXCR4 signaling pathway. LDLR and VLDLR are the smallest members of the LDLR family with a high similarity of amino acids, and both of them bound to CXCR4 as shown by the co‐IP/WB analysis. However, LDLR inhibited SDF‐1/CXCR4 without altering CXCR4 localization, whereas VLDLR activated the SDF‐1/CXCR4 signaling pathway by translocating CXCR4 from membrane to cytoplasm. In addition, LRP8 enhanced SDF‐1/CXCR4 signal transduction by stabilizing CXCR4 protein on the cell membrane. As a general ligand of the LDLR family, ApoE treatment activated SDF‐1/CXCR4 in our tested cell lines. However, because of the diversity of the LDLR family in regulating CXCR4, ApoE might also be inhibitory for CXCR4 in other cells with different a composition of LDLR family members. Precisely how the LDLR family members and their ligands such as ApoE regulate SDF‐1/CXCR4 signaling pathway is challenging and warrants further investigation. It should be interesting to identify the smallest peptides of the LDLR family members that differently regulate CXCR4.

Because CXCR4 belongs to the large GPCR family, it may be possible that the LDLR family can also physically and functionally interact with other GPCRs. Supporting this hypothesis, we recently demonstrated that LRP5/6 can directly bind to GPCR Frizzled [13] and knockdown of LRP5/6 affects multiple downstream targets of GPCRs [19]. In addition, although we focused on the regulatory effect of LDLR family members on the SDF‐1/CXCR4 signaling pathway in the present study, CXCR4 may also affect the LDLR family.

Our research primarily focuses on models of triple‐negative breast cancer (TNBC), the most aggressive subtype of breast cancer with limited treatment options. Recent studies have identified key factors involved in TNBC progression and treatment resistance. For example, GPRC5A has been linked to increased metastasis and chemotherapy resistance in TNBC [20]. Additionally, lncRNA LYPLAL1‐DT shows promise as a tumor suppressor by inhibiting TNBC cell proliferation [21]. New therapeutic approaches, such as cryoprotected nanoparticles, have also shown effectiveness in improving TNBC treatment outcomes [22]. Our work not only introduces novel methods to stratify patients for anti‐CXCR4 treatment by assessing LDLR family member expression, but also suggests a crosstalk between two protein families based on structural similarities. This may provide valuable insights for future drug design against TNBC.

In summary, the present study has revealed that the LDLR family may generally regulate CXCR4, endowing its members with an unexpected novel role of becoming new breast cancer therapy targets and prognostic markers.

Materials and methods

Cell culture

Mouse breast cancer 4T1 cell lines were obtained from Dr F. Miller in 2008 [23]. MDA‐MB‐231 cells (RRID: CVCL_0062) were obtained from Dr Z. Yu in 2017 [9]. HepG2 cells, Cos7 cells and HEK‐293 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). 4T1, MDA‐MB‐231, HepG2 (RRID: CVCL_0027), Cos7 and HEK293 (RRID: CVCL_0045) cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5 units·mL⁻¹ penicillin and 5 mg·mL⁻¹ streptomycin in a humidified incubator at 37 °C. Absence of mycoplasma infection was regularly verified using a Mycoplasma Detection kit (Bimake, Houston, TX, USA). Low‐passage cell lines were used in this study.

Co‐IP/WB analyses

Co‐IP and WB analyses were performed as previously described [9]. Total protein was extracted using NP‐40 lysis buffer (Beyotime, China). Membrane protein and cytosolic protein were extracted using membrane and cytosolic protein extraction kit (Beyotime) in accordance with the manufacturers' instructions. CM containing ApoE was produced by HEK293 cells. 4T1 and HepG2 cells were pretreated with Vector‐CM or ApoE‐CM for 4 h before SDF‐1 treatment (Fig. 4B,C). Protein band intensities from western blots were quantified using densitometry. The relative expression levels of target proteins were normalized to the intensity of loading controls. Analysis was performed using imagej (NIH, Bethesda, MD, USA) and data are presented as the fold change compared to control conditions. The results are representative of at least three independent experiments.

Cell migration assay

Transwell analysis was performed as previously described [9]. Briefly, cells were seeded onto a six‐well plate and transfected with indicated small interfering RNAs using RNAimax (Therno Fisher Scientific, Waltham, MA, USA) or transfected with the indicated plasmids using Fugene HD (Promega, Madison, WI, USA) for 48 h. Then, cells were seeded into the upper chamber of Transwell 24‐well plates with an 8‐μm polycarbonate membrane (Corning Inc., Corning, NY, USA) in serum‐free medium for cell migration assay. AMD3100 (10 μm) (Selleck, Houston, TX, USA) was added into the upper chamber. After 12 h, cells that migrated through the filter into the bottom chamber were stained using crystal violet (Sigma‐Aldrich, St Louis, MO, USA) and counted. The number of cells that underwent cell migration was normalized to control conditions and expressed as a migration index. The results are representative of three independent experiments.

Immunofluorescence analysis

HEK293 cells were fixed in 4% paraformaldehyde solution after transfection of indicated plasmids for 48 h. Then, the cells were blocked with 10% donkey serum in phosphate‐buffered saline‐Tween 20 at room temperature for 1 h, and incubated with anti‐myc antibody (Abmart, Berkeley Heights, NJ, USA) at 4 °C overnight. The cells were treated with Alexa Fluor 555 goat anti‐mouse IgG (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature, washed with phosphate‐buffered saline‐Tween 20, incubated with 300 nm 4′,6‐diamidino‐2‐phenylindole staining solution for 5 min and then examined by confocal microscopy.

Tumor implantation in mice

4T1 tumors were utilized for immunofluorescence staining. Briefly, female BALB/c mice (6–8 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and housed under specific pathogen‐free conditions under a 12 : 12 h dark/light photocycle. Mice had ad libitum access to standard rodent chow and water. In total, 1 × 10⁶ 4T1 cells were orthotopically injected into the mammary fat pads of the BALB/c mice. Tumor samples were collected before the tumor size reached 2 cm³.

Cancer patient survival analysis

Overall survival curves were generated based on the NKI295 dataset [14] and analyzed using Kaplan–Meier survival analysis coupled with a log‐rank significance test or a Gehan–‐Breslow–Wilcoxon test, as previously described [9]. For CXCR4, overall survival was analyzed between 199 high CXCR4 level and 96 low CXCR4 level patients [13]. For LDLR, overall survival was analyzed between 149 high LDLR level and 146 low LDLR level patients. For VLDLR, overall survival was analyzed between 104 high VLDLR level and 191 low VLDLR level patients. For LRP1, overall survival was analyzed between 148 high LRP1 level and 147 low LRP1 level patients. For LRP8, overall survival was analyzed between 160 high LRP8 level and 135 low LRP8 level patients. For ApoE, overall survival was analyzed between 146 high ApoE level and 149 low ApoE level patients. For SORL1, overall survival was analyzed between 147 high SORL1 level and 148 low SORL1 level patients. For LRP2, overall survival was analyzed between 148 high LRP2 level and 147 low LRP2 level patients. For LRP5, overall survival was analyzed between 147 high LRP5 level and 148 low LRP5 level patients. For LRP1B, overall survival was analyzed between 121 high LRP1B level and 174 low LRP1B level patients. For LRP4, overall survival was analyzed between 146 high LRP4 level and 149 low LRP4 level patients.

Ethical declaration

All animal studies have been approved by the Institutional Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine, China (Approval No: FJTCM IACUC 2022–233), comply with the ARRIVE guidelines, and were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. For cancer patient survival analysis, no human samples were used, and all data were generated based on the NKI295 dataset.

Statistical analysis

All statistical analyses were performed using SPSS, version 22.0 (IBM Corp., Armonk, NY, USA) as previously described [24] and data are presented as the mean ± SD. One‐way analysis of variance with Bonferroni's post‐hoc analysis was used for comparisons between three or more groups and an independent sample t‐test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Author contributions

JZ performed most of the experiments and the initial writing of the manuscript. JC assisted in performing the experiments. DW, EM and HY contributed to new reagents/analytical tools and critical discussion. WZ wrote the manuscript. The manuscript was revised by JP and DnR. WZ conceived of and supervised the entire project. All authors read and approved the final version of the manuscript submitted for publication.

Peer review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1111/febs.70016.

Supporting information

Fig. S1. Photo images and quantification of transwell analysis of cell migration following transfection of LDLR with or without treatment of SDF‐1 in MDA‐MB‐231 cells.

Fig. S2. Localization of endogenous LDLR and CXCR4 in murine 4T1 tumor.

Fig. S3. Structural analysis of CXCR4 and indicated receptors as predicted by AlphaFold 3, including complexes with mouse and human LDLR, VLDLR, LRP1, LRP8 and APOE.

FEBS-292-4580-s001.pdf^{(30MB, pdf)}

Acknowledgements

We thank Dr F. Miller for providing 4T1 cells. This work was supported by Youth Science and Technology Innovation Talent Cultivation Program of FJTCM (XQB202201), Scientific Research Foundation for the High‐level Talents, Fujian University of Traditional Chinese Medicine (X2019001‐talent, X2021001‐talent, X2021002‐talent, X2021003‐talent).

Contributor Information

Dan‐ni Ren, Email: danny1217@163.com.

Weidong Zhu, Email: wzhu@tongji.edu.cn.

Data availability statement

The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.

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7. Hernandez‐Lopez C, Varas A, Sacedon R, Jimenez E, Munoz JJ, Zapata AG & Vicente A (2002) Stromal cell‐derived factor 1/CXCR4 signaling is critical for early human T‐cell development. Blood 99, 546–554. [DOI] [PubMed] [Google Scholar]
8. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, Bronson RT & Springer TA (1998) Impaired B‐lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4‐ and SDF‐1‐deficient mice. Proc Natl Acad Sci USA 95, 9448–9453. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Zhang J, Chen J, Wo D, Yan H, Liu P, Ma E, Li L, Zheng L, Chen D, Yu Z et al. (2019) LRP6 ectodomain prevents SDF‐1/CXCR4‐induced breast cancer metastasis to lung. Clin Cancer Res 25, 4832–4845. [DOI] [PubMed] [Google Scholar]
10. Tamai K, Semenov M, Kato Y, Spokony R, Liu C, Katsuyama Y, Hess F, Saint‐Jeannet JP & He X (2000) LDL‐receptor‐related proteins in Wnt signal transduction. Nature 407, 530–535. [DOI] [PubMed] [Google Scholar]
11. Brown SD, Twells RC, Hey PJ, Cox RD, Levy ER, Soderman AR, Metzker ML, Caskey CT, Todd JA & Hess JF (1998) Isolation and characterization of LRP6, a novel member of the low density lipoprotein receptor gene family. Biochem Biophys Res Commun 248, 879–888. [DOI] [PubMed] [Google Scholar]
12. Go GW & Mani A (2012) Low‐density lipoprotein receptor (LDLR) family orchestrates cholesterol homeostasis. Yale J Biol Med 85, 19–28. [PMC free article] [PubMed] [Google Scholar]
13. Ren DN, Chen J, Li Z, Yan H, Yin Y, Wo D, Zhang J, Ao L, Chen B, Ito TK et al. (2015) LRP5/6 directly bind to frizzled and prevent frizzled‐regulated tumour metastasis. Nat Commun 6, 6906. [DOI] [PubMed] [Google Scholar]
14. van de Vijver MJ, He YD, van't Veer LJ, Dai H, Hart AAM, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ et al. (2002) A gene‐expression signature as a predictor of survival in breast cancer. N Engl J Med 347, 1999–2009. [DOI] [PubMed] [Google Scholar]
15. Willnow TE, Moehring JM, Inocencio NM, Moehring TJ & Herz J (1996) The low‐density‐lipoprotein receptor‐related protein (LRP) is processed by furin in vivo and in vitro. Biochem J 313(Pt 1), 71–76. [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Lane‐Donovan C & Herz J (2017) ApoE, ApoE receptors, and the synapse in Alzheimer's disease. Trends Endocrinol Metab 28, 273–284. [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Xian X, Pohlkamp T, Durakoglugil MS, Wong CH, Beck JK, Lane‐Donovan C, Plattner F & Herz J (2018) Reversal of ApoE4‐induced recycling block as a novel prevention approach for Alzheimer's disease. eLife 7, e40048. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J et al. (2024) Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Wo D, Peng J, Ren DN, Qiu L, Chen J, Zhu Y, Yan Y, Yan H, Wu J, Ma E et al. (2016) Opposing roles of Wnt inhibitors IGFBP‐4 and Dkk1 in cardiac ischemia by differential targeting of LRP5/6 and beta‐catenin. Circulation 134, 1991–2007. [DOI] [PubMed] [Google Scholar]
20. Ou X, Tan Y, Xie J, Yuan J, Deng X, Shao R, Song C, Cao X, Xie X, He R et al. (2024) Methylation of GPRC5A promotes liver metastasis and docetaxel resistance through activating mTOR signaling pathway in triple negative breast cancer. Drug Resist Updat 73, 101063. [DOI] [PubMed] [Google Scholar]
21. Tang Y, Tian W, Zheng S, Zou Y, Xie J, Zhang J, Li X, Sun Y, Lan J, Li N et al. (2023) Dissection of FOXO1‐induced LYPLAL1‐DT impeding triple‐negative breast cancer progression via mediating hnRNPK/beta‐catenin complex. Res Wash DC 6, 0289. [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Xu C, Zhang C, Ganesan K, Chen Q, Tang H, Gao F, Liu Q, Wu J, Sui Y, Li P et al. (2023) Anti‐migratory properties of cryoprotective Isoliquiritigenin‐zein phosphatidylcholine nanoparticles prevent triple‐negative breast cancer through PI3K‐mTOR and MMP2/9 pathways. Curr Med Chem 31, 0945. [DOI] [PubMed] [Google Scholar]
23. Aslakson CJ & Miller FR (1992) Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 52, 1399–1405. [PubMed] [Google Scholar]
24. Zhu X, Ma E, Ge Y, Yuan M, Guo X, Peng J, Zhu W, Ren DN & Wo D (2024) Resveratrol protects against myocardial ischemic injury in obese mice via activating SIRT3/FOXO3a signaling pathway and restoring redox homeostasis. Biomed Pharmacother 174, 116476. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Photo images and quantification of transwell analysis of cell migration following transfection of LDLR with or without treatment of SDF‐1 in MDA‐MB‐231 cells.

Fig. S2. Localization of endogenous LDLR and CXCR4 in murine 4T1 tumor.

Fig. S3. Structural analysis of CXCR4 and indicated receptors as predicted by AlphaFold 3, including complexes with mouse and human LDLR, VLDLR, LRP1, LRP8 and APOE.

FEBS-292-4580-s001.pdf^{(30MB, pdf)}

Data Availability Statement

The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.

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[febs70016-bib-0009] 9. Zhang J, Chen J, Wo D, Yan H, Liu P, Ma E, Li L, Zheng L, Chen D, Yu Z et al. (2019) LRP6 ectodomain prevents SDF‐1/CXCR4‐induced breast cancer metastasis to lung. Clin Cancer Res 25, 4832–4845. [DOI] [PubMed] [Google Scholar]

[febs70016-bib-0010] 10. Tamai K, Semenov M, Kato Y, Spokony R, Liu C, Katsuyama Y, Hess F, Saint‐Jeannet JP & He X (2000) LDL‐receptor‐related proteins in Wnt signal transduction. Nature 407, 530–535. [DOI] [PubMed] [Google Scholar]

[febs70016-bib-0011] 11. Brown SD, Twells RC, Hey PJ, Cox RD, Levy ER, Soderman AR, Metzker ML, Caskey CT, Todd JA & Hess JF (1998) Isolation and characterization of LRP6, a novel member of the low density lipoprotein receptor gene family. Biochem Biophys Res Commun 248, 879–888. [DOI] [PubMed] [Google Scholar]

[febs70016-bib-0012] 12. Go GW & Mani A (2012) Low‐density lipoprotein receptor (LDLR) family orchestrates cholesterol homeostasis. Yale J Biol Med 85, 19–28. [PMC free article] [PubMed] [Google Scholar]

[febs70016-bib-0013] 13. Ren DN, Chen J, Li Z, Yan H, Yin Y, Wo D, Zhang J, Ao L, Chen B, Ito TK et al. (2015) LRP5/6 directly bind to frizzled and prevent frizzled‐regulated tumour metastasis. Nat Commun 6, 6906. [DOI] [PubMed] [Google Scholar]

[febs70016-bib-0014] 14. van de Vijver MJ, He YD, van't Veer LJ, Dai H, Hart AAM, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ et al. (2002) A gene‐expression signature as a predictor of survival in breast cancer. N Engl J Med 347, 1999–2009. [DOI] [PubMed] [Google Scholar]

[febs70016-bib-0015] 15. Willnow TE, Moehring JM, Inocencio NM, Moehring TJ & Herz J (1996) The low‐density‐lipoprotein receptor‐related protein (LRP) is processed by furin in vivo and in vitro. Biochem J 313(Pt 1), 71–76. [DOI] [PMC free article] [PubMed] [Google Scholar]

[febs70016-bib-0016] 16. Lane‐Donovan C & Herz J (2017) ApoE, ApoE receptors, and the synapse in Alzheimer's disease. Trends Endocrinol Metab 28, 273–284. [DOI] [PMC free article] [PubMed] [Google Scholar]

[febs70016-bib-0017] 17. Xian X, Pohlkamp T, Durakoglugil MS, Wong CH, Beck JK, Lane‐Donovan C, Plattner F & Herz J (2018) Reversal of ApoE4‐induced recycling block as a novel prevention approach for Alzheimer's disease. eLife 7, e40048. [DOI] [PMC free article] [PubMed] [Google Scholar]

[febs70016-bib-0018] 18. Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard AJ, Bambrick J et al. (2024) Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500. [DOI] [PMC free article] [PubMed] [Google Scholar]

[febs70016-bib-0019] 19. Wo D, Peng J, Ren DN, Qiu L, Chen J, Zhu Y, Yan Y, Yan H, Wu J, Ma E et al. (2016) Opposing roles of Wnt inhibitors IGFBP‐4 and Dkk1 in cardiac ischemia by differential targeting of LRP5/6 and beta‐catenin. Circulation 134, 1991–2007. [DOI] [PubMed] [Google Scholar]

[febs70016-bib-0020] 20. Ou X, Tan Y, Xie J, Yuan J, Deng X, Shao R, Song C, Cao X, Xie X, He R et al. (2024) Methylation of GPRC5A promotes liver metastasis and docetaxel resistance through activating mTOR signaling pathway in triple negative breast cancer. Drug Resist Updat 73, 101063. [DOI] [PubMed] [Google Scholar]

[febs70016-bib-0021] 21. Tang Y, Tian W, Zheng S, Zou Y, Xie J, Zhang J, Li X, Sun Y, Lan J, Li N et al. (2023) Dissection of FOXO1‐induced LYPLAL1‐DT impeding triple‐negative breast cancer progression via mediating hnRNPK/beta‐catenin complex. Res Wash DC 6, 0289. [DOI] [PMC free article] [PubMed] [Google Scholar]

[febs70016-bib-0022] 22. Xu C, Zhang C, Ganesan K, Chen Q, Tang H, Gao F, Liu Q, Wu J, Sui Y, Li P et al. (2023) Anti‐migratory properties of cryoprotective Isoliquiritigenin‐zein phosphatidylcholine nanoparticles prevent triple‐negative breast cancer through PI3K‐mTOR and MMP2/9 pathways. Curr Med Chem 31, 0945. [DOI] [PubMed] [Google Scholar]

[febs70016-bib-0023] 23. Aslakson CJ & Miller FR (1992) Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 52, 1399–1405. [PubMed] [Google Scholar]

[febs70016-bib-0024] 24. Zhu X, Ma E, Ge Y, Yuan M, Guo X, Peng J, Zhu W, Ren DN & Wo D (2024) Resveratrol protects against myocardial ischemic injury in obese mice via activating SIRT3/FOXO3a signaling pathway and restoring redox homeostasis. Biomed Pharmacother 174, 116476. [DOI] [PubMed] [Google Scholar]

PERMALINK

Physical and functional interactions between LDLR family members and CXCR4 in breast cancer

Jiankang Zhang

Jinxiao Chen

Da Wo

En Ma

Hongwei Yan

Jun Peng

Dan‐ni Ren

Weidong Zhu

Abstract

Abbreviations

Introduction

Fig. 1.

Results

LDLR and VLDLR interact with CXCR4 and influence the SDF‐1/CXCR4 signaling pathway

LRP1 and LRP8 modulate the SDF‐1/CXCR4 signaling pathway

Fig. 2.

Impact of other LDLR family members on SDF‐1/CXCR4 prognosis in breast cancer patients

Fig. 3.

Apolipoprotein E (ApoE) as a ligand of the LDLR family influences the SDF‐1/CXCR4 signaling pathway

Fig. 4.

Discussion

Materials and methods

Cell culture

Co‐IP/WB analyses

Cell migration assay

Immunofluorescence analysis

Tumor implantation in mice

Cancer patient survival analysis

Ethical declaration

Statistical analysis

Conflicts of interest

Author contributions

Peer review

Supporting information

Acknowledgements

Contributor Information

Data availability statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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