MiR-651-3p/CAV1/EGFR axis Modulates Progression and Vasculogenic Mimicry in Triple-negative Breast Cancer

doi:10.21203/rs.3.rs-2490031/v1

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MiR-651-3p/CAV1/EGFR axis Modulates Progression and Vasculogenic Mimicry in Triple-negative Breast Cancer

https://doi.org/10.21203/rs.3.rs-2490031/v1

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Background: Breast cancer (BC) has become the most common type of cancer and the second most common cause of cancer-related death. In comparison with other subtypes of breast cancer, triple-negative breast cancer (TNBC) is highly aggressive, more likely to metastasize, and has a shorter survival time. MiRNAs play an inhibitory or promoting role in cancer, and are involved in several cell signaling pathways, including growth, proliferation, differentiation, and survival. Vasculogenic mimicry (VM) is associated with invasive disease, tumor spread, metastasis, and poor prognosis. Additional research is needed to determine the mechanisms governing VM formation in TNBC.

Methods: We measured RNA and protein expression using quantitative real-time PCR (RT-qPCR) and western blotting. Assays assessing cell proliferation were conducted with CCK-8, cell cycle, and colony formation. Cell migration and invasion were evaluated using transwells, scratch tests, and high-intensity imaging. Luciferase reporter assays were used to confirm miR-651-3p and CAV1 target relationships.

Results: In TNBC, miR-651-3p was significantly overexpressed, implicating it as an oncogene. By inhibiting CAV1 transcriptional synthesis, miR-651-3p can enhance the activity of EGFR pathways, leading to promotion of TNBC proliferation, VM formation, and migration.

Conclusion: It was determined that miR-651-3p/CAV1/EGFR axis could be a therapeutic target for TNBC in this study.

Triple-negative breast cancer

miR-651-3p

CAV1

Among women, breast cancer (BC) is the most common type of cancer and the second most common cause of cancer-related death.^[1] Global cancer statistics show that BC alone accounted for 30% of cancers in females and an estimated 281550 new cases and more than 43600 deaths occurred in 2021.^[2] BC is a highly heterogeneous cancer type with different treatment modalities and prognoses among patients. A triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2).^[3–6] TNBC is most common in young premenopausal women under the age of 40, who make up about 15–20% of all breast cancer patients.^[7] In comparison with other subtypes of breast cancer, TNBC is highly aggressive, more likely to metastasize, and has a shorter survival time.^[8] TNBC has a specific molecular phenotype that makes it difficult to benefit from endocrine therapy or molecular targeted therapy, while the efficacy of postoperative adjuvant chemoradiotherapy is low, and the current dilemma is postoperative recurrence.^[9] Even though breast cancer treatment has advanced in leaps and bounds in recent years, oncologists still face a significant challenge in preventing disease recurrence and metastasis. There is an urgent need to develop new treatment options and targets to combat TNBC, as it poses a threat to global health.

Angiogenesis regulates tumor growth, invasion, and metastasis by providing oxygen and nutrients to the tumor.^[10] Several studies have shown that VEGF ^[11] is an important target for anti-angiogenic drugs.^[10] Despite the fact that angiogenesis inhibitors have shown good efficacy in some solid tumors, breast cancer patients have not yet benefited from these drugs in terms of survival. ^{[12, 13]} In spite of the fact that angiogenesis plays an important role in the growth of tumor blood vessels, there are other mechanisms that are capable of meeting tumor oxygen and nutrient requirements.^[14] Interestingly, in 1999, Hendricks^[15] proposed a novel model of tumor blood supply, Vasculogenic mimicry (VM) formed by tumor cells rather than endothelial cells. In addition to acquiring malignant phenotypes during VM formation, cancer cells also increase the expression of numerous vascular-related markers.^[16] The mechanisms governing VM formation in TNBC remain unclear, and additional research is necessary.

MicroRNAs (miRNAs) are a class of non-coding, single-stranded RNAs that have 18–25 nucleotides.^[17] By binding to its specific seed sequence, the miRNA preferentially binds to the 3'-UTR of the target mRNA. In some cases, it can also bind to the 5'-UTR and coding sequence of the mRNA, resulting in mRNA degradation or blocking translation.^[18–20] It has been estimated that approximately 60% of human genes are reported to contain multiple seed binding sites.^{[21, 22]} There is a complex role for miRNAs in regulating gene expression, and they are involved in a variety of cell signaling pathways, including growth, proliferation, differentiation, and survival, as well as playing an inhibitory or promoting role in cancer.^[23–26] The relationship between miRNAs and BC was first established by scholars in 2005, and several subsequent studies have demonstrated that miRNAs can be used not only to determine the stage of BC and to predict patient survival, but also as biomarkers to aid in BC diagnosis and prognosis.^[27] According to recent studies, miR-651 plays a role in the progression of non-small cell lung cancer (NSCLC) and nasopharyngeal carcinoma.^{[28, 29]} Among the several miRNAs known, the relationship between miR-651-3p and TNBC remains unclear. A thorough investigation of specific mechanisms is urgently required.

As a key component of caveolae, which are cave-shaped invaginated structures of the plasma membrane, caveolin-1 (CAV1) is a cell membrane intrinsic protein with a molecular weight of 21–24 KDa. Endocytosis, extracellular matrix organization, cholesterol distribution, cell migration, and signaling are strongly associated with caveolin-1 (CAV1).^[30–33] It should be noted that CAV1 is generally reported to play a paradoxical role ranging from tumor suppression to tumor initiation, depending on the subtype and stage of BC.^[34–38] Several studies have shown that CAV1 plays an important role in tumor progression by activating EGFR and inducing endocytosis in cancer.^{[39, 40]} As CAV1 plays a complex role in tumorigenesis and development, clarifying its key mechanisms is essential for the diagnosis and treatment of BC.

Using clinical TNBC tissue samples, we examined miR-651-3p expression levels and functions for the first time. Furthermore, we investigated the mechanical interplay between miR-651-3p, CAV1 and EGFR pathways in the progression and formation of VM in TNBC. A novel insight into the treatment of TNBC may be gained as a result of this research.

Bioinformatics Analysis

The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) provided the mRNA expression data for CAV1. UALCAN website (http://ualcan.path.uab.edu/analysis.html ) was employed to detect the differential expression levels of CAV1 mRNA in breast cancer. The STRING database (https://string-db.org/) was employed to construct a protein-protein interaction (PPI) network. R software was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using the clusterProfiler package. Using target prediction algorithms databases, such as miRDB and TargetScan, we searched for targets of has-miR-651-3p.

Human Tissue

We obtained fresh BC tissues and paired adjacent noncancerous tissues from patients who underwent surgery at Tianjin Medical University Cancer Institute & Hospital (Tianjin, China) without receiving chemotherapy and radiotherapy beforehand, and each pathological diagnosis was confirmed following surgery. The tumor tissues are separated from the adjacent non-cancerous tissues by at least 2 cm. Within two hours of surgery, tissues were immediately frozen in liquid nitrogen for 10–20 minutes, and then stored at -80°C for future analysis. The collection of discarded human tissues was approved by the Research Ethics Board of Tianjin Medical University Cancer Institute & Hospital. Written informed consent was provided by all patients involved in this study. The study design and conduct complied with all relevant regulations regarding the use of human study participants and was conducted in accordance with the criteria set by the Declaration of Helsinki.

Cell Culture

Human BC cell lines MDA-MB-231, BT-549 and HCC-1806, as well as MCF-10A, HEK-293T cells, were purchased from the Shanghai Institute of Cell Biology of the Chinese Academy of Sciences (Shanghai, China), cultured in DMEM/ RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Incubation of all of these cell lines was conducted in a humidified incubator at 37°C with 5% CO2.

Cell transfection

Using Lipofectamine 3000 (Invitrogen) and Opti-MEM (Gibco, USA), cells were seeded into 6-well plates (approximately 3×10⁶ cells per well) and transfected when confluency reached 50% using the nucleotides or plasmids mentioned above. In this study, CAV1 knockdown and overexpression plasmids were purchased from RiboBio (Guangzhou, China) as well as miR-651-3p mimics and miR-651-3p inhibitors.

Immunofluorescence

Cells were seeded on glass coverslips in 24-well plates, washed with PBS three times, fixed with 4% paraformaldehyde for 10 minutes, permeabilized and blocked with 5% BSA and 0.1% Triton X-100 in PBS solution for 1 hour at 37°C. After incubating the glass coverslips with primary antibodies overnight at 4°C, the coverslips were washed in PBS. Afterwards, the secondary antibody was incubated at 37°C for 1 hour, followed by 10 minutes of staining with DAPI (Sigma). Finally, a fluorescence microscope was used to observe it.

RNA Extraction and Quantitative Real-Time PCR (RT-qPCR)

The total RNA from the cultures and tissues was extracted by the TRIzol (Invitrogen) method according to the manufacturer’s instructions, and the RNA purity and concentration were measured using a UV spectrophotometer. Using a reverse transcription kit (YEASEN, Shanghai, China), total RNA (500ng-1000ng) was reverse transcribed into cDNA. With the cDNA as a template, the qPCR system was prepared according to the instruction of the StarLighter HP SYBR Green qPCR Mix (FOREVER STAR, Beijing, China). Data were processed and calculated based on the 2^−ΔΔCt method. U6 snRNA was used as an internal control for the miRNAs; β-actin, for the mRNA levels. The primer sequences utilized in our research were shown in Table 1.

Table 1

The primer sequences utilized in our research.
miR-651-3p	Forward Primer: 5’- CGCGGCCAAAGGAAAGTGTATC-3’ Reverse Primer: 5- ATCCAGTGCAGGGTCCGAGG-3’
U6	Forward Primer: 5’- CTCGCTTCGGCAGCACA-3’ Reverse Primer: 5- AACGCTTCACGAATTTGCGT-3’
CAV1	Forward Primer: 5’- GCGACCCTAAACACCTCAAC-3’ Reverse Primer: 5- ATGCCGTCAAAACTGTGTGTC-3’
β-actin	Forward Primer: 5’- CATGTACGTTGCTATCCAGGC-3’ Reverse Primer: 5- CTCCTTAATGTCACGCACGAT-3’

Dual-Luciferase Reporter Assay

The fragment of wild-type (WT) or mutant (MUT) CAV1 3′UTR was cloned into a pmirGLO vector plasmid (Promega, USA). The HEK-293T cells (3×10⁵ cells per well) were seeded in 24 wells and co-transfected with CAV1-3’UTR-WT or CAV1-3’UTR-MUT and miR-651-3p mimics or mimics control into the cells. The activity of luciferase was detected 48 hours after transfection using a dual luciferase reporter system (YEASEN, Shanghai, China), and the ratio was calculated and normalized to that of Renilla luciferase.

Cell Proliferation Assay

Cell proliferation was measured using Cell Counting Kit-8 solution (CCK8) (YEASEN, Shanghai, China). A 96-well plate was used to collect cells from each group at a density of 2.5×103 cells/100ul/well. More than 3 duplicate wells were set in the group. Incubate each well in an incubator for three hours after replacing the cultured medium with fresh medium containing 10% CCK8. Microplate readers were used to detect OD values at 450 nm.

Colony Formation Assay

Colony formation assays were performed by seeding 500 cells per well in a 6-well culture plate after transfection for 24-48h and incubated at 37℃ for 10–14 days. When visible colonies appeared on the plate, the culture medium was discarded, washed three times with sterile PBS, and then fixed with formaldehyde for 20 minutes at -20°C and stained with crystal violet solution for 15 minutes. The dye was washed off with running water and dried in the air after being washed off with running water.

Cell Migration Assay

The migratory capacity of the cells was determined by using Transwell chambers (Corning, Shanghai, China), cell scratch tests, and high intensity imaging (PerkinElmer, PE operetta CLS, USA) migration assays. The lower chamber of the transwell chamber was filled with 400 ul of medium containing 10% FBS. Then, 2×10³ cells in 200 ul serum-free DMEM or RPMI-1640 medium were transferred to the transwell upper chamber. Using a cotton swab, the non-migrated cells from the upper chamber were gently removed and washed with sterile PBS, before the chamber was fixed with formaldehyde for 20 minutes and stained with 1% crystal violet for 15 minutes. Cells were seeded in 6-well plates for the cell scratch test. A 20 ul pipette tip was used to gently scratch each cell after 90% fusion, then the cells were washed three times with PBS before being added to medium without FBS. At the time points of 0 hours and 24 hours, the width of the scratch was observed and photographed.

Cell Cycle Analysis

We digested each well, collected the cells, and washed them three times with cold PBS. The cells were then fixed in 3ml of 75% chilled ethanol overnight at -20°C. The next day, centrifuge at 1500 rpm for 5 minutes to remove ethanol, and then wash three times with cold PBS to remove any remaining ethanol. The cells were then stained with 500 ul propidium iodide (PI) solution (BD Biosciences, USA) at 4℃ for 30 minutes. The stained samples were tested and analyzed by ModFit (Verity Software House, Topsham, ME, USA).

Vasculogenic Mimicry Formation Assays

BC cells were tested in vitro for their ability to form VM by using a three-dimensional cell culture method. It was coated with 50 ul/well Matrigel (BD, USA) and cured at 37°C for 30 minutes. During incubation at 37°C for 6 hours, the cells were trypsinized and suspended in complete medium at a density of 4×10⁵ cells per ml, seeded 50 ul suspensions onto the surface of Matrigel, and incubated for 6 hours. A calculation was made to determine the number of mature tubes. A light microscope (20X) was used to take the images.

Western Blot Analysis

Cells and tissues were lysed using RIPA lysate with protease PMSF (100:1). Total protein (30–50 ug) was separated on 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to 0.45 µm PVDF membrane (Millipore, USA). For blocking purposes, the membranes were blocked with 5% nonfat milk (BD, USA) for 1 hour at room temperature, followed by overnight incubation at 4°C with the corresponding primary antibody. Following incubation with primary antibodies, membranes were washed three times with TBST, then incubated for one hour at room temperature with secondary antibodies, and then washed three times with TBST. Lastly, enhanced chemiluminescence reagents (ECL) (Millipore, USA) were used to visualize the bands on the membranes.

Statistical analysis

SPSS 26.0 statistical software was used to calculate all statistical analyses. All data in this research were presented as mean values ± standard deviation (SD) of at least 3 independent experiments. An analysis of differences between groups was conducted using Student's t-test. The significance was represented by * p < 0.05, **p < 0.01, ***p < 0.001.

miR-651-3p is dysregulated in TNBC tissues and cell lines

A qPCR study was conducted to validate the expression profiles of miR-651-3p in 34 paired TNBC tissues, including tumor tissues and noncancerous adjacent tissues, in order to determine whether miR-651-3p may have carcinogenic potential in TNBC. As a result of the qPCR analysis, it was found that miR-651-3p expression was higher in tumor tissues than in adjacent tissues (Fig. 1A). In addition, we observed a reduction in miR-651-3p expression in TNBC cell lines as compared to normal breast epithelial cells (Fig. 1B).

miR-651-3p regulates cell proliferation, migration, and VM formation of TNBC

It has already been found that miR-651-3p is aberrantly expressed in TNBC; therefore, the following assays were performed in order to clarify whether miR-651-3p is causing a gain-of-function effect on the biological functions of TNBC cells. Firstly, the miR-651-3p mimics and inhibitors were transfected to investigate the subsequent functional assays (Fig. 1C). Transwell experiments revealed that miR-651-3p mimics significantly enhanced the migratory ability of TNBC cells, whereas miR-651-3p inhibitors significantly reduced it (Fig. 1D). To determine the influence of miR-651-3p on cell proliferation, the CCK8 experiment and flow cell cycle were performed. As shown in Fig. 1E, compared with the control group, miR-651-3p mimics promoted the proliferation of TNBC cells, while inhibiting miR-651-3p suppressed the proliferation. Furthermore, cell cycle analysis analyzed that TNBC cells were arrested in G0/G1 phase in the miR-651-3p inhibitors group, whereas the mimics group included more cells entering the S phase (Fig. 1G, H). According to the above data, miR-651-3p may contribute to the proliferation of TNBC cells.

VM is fluid conduction channels formed by highly aggressive and genetically dysregulated tumor cells that serve as complementary sources of blood and nutrient supply.^[41] In order to examine whether miR-651-3p affects the formation of VM networks in TNBC, the VM formation assay was used. It was found that the VM formation ability was significantly inhibited in the miR-651-3p inhibitor group compared with the mimic group (Fig. 1F). These data revealed that miR-651-3p may act as an oncogene in TNBC.

CAV1 served as a direct target gene of miR-651-3p

As a key component of non-coding RNA, miRNAs are known to regulate epigenetic changes in cancer.^[42] To further explore the downstream targets of miR-651-3p in TNBC, we used TargetScan and miRDB to identify the targets. In our analysis of the top 50 gene sets from two databases, we identified two candidates, AMMECR1 and CAV1, after taking into account the overlap between the target molecules. (Fig. 2A) Afterwards, comprehensive bioinformatic analyses revealed that the expression of CAV1 was reduced in tumor tissues, whereas AMMECR1 showed the opposite pattern. (Fig. 2B).

According to our previous experiments, miR-651-3p is expressed at a higher level in cancer tissues than in normal tissues, indicating that CAV1 is more likely to function as a downstream target factor of miR-651-3p in the progression of TNBC. Our hypothesis was also confirmed by the prediction of binding site information. According to Fig. 2C, the 3'UTR of CAV1 mRNA can be directly regulated by miR-651-3p in a highly conserved manner across species by miR-651-3p. In Western blot assays, the expression of CAV1 protein decreased after transfection with miR-651-3p mimics, while it increased after transfection with miR-651-3p inhibitors (Fig. 2D). As a follow-up, dual-luciferase reporter assays were conducted to confirm this regulation. They demonstrated that luciferase activity was significantly increased when miR-651-3p mimics were co-transfected with WT-CAV1-3'UTR, but not with MUT-CAV1-3'UTR (Fig. 2E). The results of these experiments strongly support our hypothesis that miR-651-3p and CAV1 are related through targeting.

CAV1 is aberrantly downregulated in pan-cancer tissues and correlates with disease progression

Recent research has focused on the role of CAV1 in the progression of human tumors, such as pancreatic cancer, lung cancer, and breast cancer.^[43–46] It is noteworthy that CAV1 plays dual roles in BC as demonstrated in recent studies.^[47] Therefore, it is necessary to explore further the role of CAV1 in TNBC. As a first step, we examined the expression of CAV1 in pan-cancer using a public dataset. In most cancer species, radar charts showed that CAV1 mRNA expression levels were significantly different between cancer tissues and normal tissues(Fig. 3A, B). As shown in Fig. 3C, the expression level of CAV1 mRNA was higher in cancer tissues compared with normal tissues in BC. Additionally, low expression of CAV1 was associated with a higher pathologic stage (Fig. 3D). We also founded that with the increase of age group, the expression level of CAV1 showed a decreasing trend (Fig. 3E). By comparing the expression level of CAV1 in different molecular types of BC, we founded that TNBC has the lowest expression level among four common molecular types (Fig. 3F). To investigate the prognostic significance of CAV1 in BC, we analyzed the survival data of BC patients. As shown in Fig. 3G, low CAV1 expression group was associated with shorter OS. To verify the expression level of CAV1 and its relationship with miR-651-3p in clinical TNBC tissues, we performed qRT-PCR to evaluate the expression of CAV1 in 34 matched pairs of TNBC and adjacent non-cancer tissues to further investigate the expression level and significance of CAV1. As illustrated in Fig. 3H, the panel of CAV1 mRNA in TNBC tissues is differentially expressed between tumor and paired adjacent normal tissues. Additionally, miR-651-3p expression in the tissues was negatively correlated with CAV1. These results confirmed that the CAV1 plays a more prominent role in TNBC as a tumor suppressor.

CAV1 Regulates TNBC cell proliferation, migration, and VM formation

Initially, we established CAV1-silenced (MDA-MB-231) and CAV1-overexpressing (BT-549) cell lines and validated their expression levels by Western blot and immunofluorescence (Fig. 4A, B, C). The migration capacity of cells was determined by combining the cell scratch test, transwell assay, and high-intension imaging cell analysis in our study. Cell growth assays indicated that CAV1 knockdown promoted the proliferation of MDA-MB-231 cells, while overexpression of CAV1 impaired the proliferation of BT549 cells (Fig. 4D and 4E). Similarly, both scratch assays and transwell assays indicated that overexpression of CAV1 retarded the migration and invasion of BT549 cells, whereas inhibiting CAV1 enhanced migration and invasion of MDA-MB-231 cells (Fig. 4F,4G,4H). Further, we found that CAV1 expression significantly impacted the ability of TNBC cells to clone (Fig. 4I).

There is evidence that VM mediated by CAV1 is associated with tumor malignancy and prognosis in patients with glioma^{[48, 49]}. There is, however, no clear understanding of the mechanism by which CAV1 regulates VM in TNBC. As demonstrated by the VM assays, the expression level of CAV1 significantly affects the ability of TNBC cells to form tubules (Fig. 5A). According to the cell cycle analysis results, more cells entered the S phase after CAV1 downregulation, and more cells were arrested in the G0/G1 phase after CAV1 upregulation (Fig. 5B, C). Based on these observations, it can be concluded that CAV1 participates in the progression of TNBC.

MiR-651-3p regulates the EGFR signaling pathways by targeting CAV1

Based on the DAVID and STRING databases, we constructed a network to identify the biological function, the protein-protein interaction (PPI), and the co-expressed genes of CAV1. Figure 6A shows the PPI structure of 50 CAV1-related genes selected using the STRING tool after visualization by Cytoscape tool. Then the CytoHubba plug-in of Cytoscape was used to identify important genes in PPI by identifying the network characteristics of nodes and measuring nodes (Fig. 6B). It is obviously that CAV1 and EGFR are at the core of the complex protein interaction network, which suggests that there may be some correlation between CAV1 and EGFR.

Furthermore, KEGG pathway enrichment and GO analysis of CAV1-related molecules were also conducted. The EGFR is a type of transmembrane glycoprotein that has PTK activity (protein tyrosine kinase). Upon binding to epidermal growth factor (EGF), kinase-active homodimers or heterodimers are formed.^[50] As a result of overactivation of EGFR, abnormal enhancements of cell biology can be induced, resulting in the overactivation of downstream signaling pathways, including protection against apoptosis, angiogenesis, and VM.^[51–54] The effect of EGFR on the formation of VM in breast cancer has not been investigated, although it mediates various tumor-related signaling pathways. Thus, when functional enrichment of CAV1-related genes is being carried out, we should focus on pathways related to EGFR. According to our study, the most significant pathways associated with 38 CAV1-related genes are the PI3K-Akt signaling pathway, the MAPK signaling pathway, and the Ras signaling pathway (Fig. 7A). Based on the enrichment results, the GO data for BP (Biological process), CC (Cellular component), and MF (Molecular function) are shown in (Fig. 7B-D). Furthermore, western blot showed that downregulated or overexpressed CAV1 significantly influences the relative activation levels of EGFR and its downstream pathways (Fig. 7E).

After transfecting cells with miR-651-3p mimics or inhibitors, western blotting was performed to determine whether miR-651-3p regulates EGFR signaling through CAV1. After being transfected with miR-651-3p mimics, EGFR, AKT, MEK, and STAT3 expression was significantly activated, as shown in Fig. 7F. In contrast, after being transfected with miR-651-3p inhibitors, the EGFR signaling pathways were clearly inhibited. Additionally, miR-651-3p mimics significantly antagonized CAV1-induced activation of EGFR pathways, suggesting that miR-651-3p mimics could enhance EGFR pathways by silencing CAV1 (Fig. 7G). In order to examine the role of EGFR in VM formation, TNBC cell lines were treated with EGF and AG1478 (an inhibitor of EGFR tyrosine kinase). In comparison with NC cells, when the cells were stimulated with EGF, their tube formation capacity was enhanced, while after treatment with AG1478, the tube formation was inhibited (Fig. 7H).

CAV1 overexpression resisted miR-651-3p-induced enhancement of TNBC proliferation, migration, and VM

We performed rescue experiments in TNBC cells by overexpressing CAV1 and transfecting them with miR-651-3p mimics. As shown in Fig. 8A and 8B, CAV1 was able to reverse the facilitation effect of miR-651-3p on cell growth and migration. Interestingly, we also observed that co-transfection of miR-651-3p mimics with CAV1 inhibited the ability of miR-651-3p mimics to promote VM formation (Fig. 8C). The data presented here strongly support our hypothesis that the miR-651-3p/CAV1/EGFR axis regulates the VM formation, proliferation, and migration.

Consequently, miR-651-3p induces EGFR expression by reducing CAV1, thus activating downstream pathways of EGFR that modulate TNBC progression and VM, which are expected to be therapeutic targets. (Fig. 9)

BC is one of the most prevalent types of cancer and has become a serious threat to human health.^[1] Detection rates of early-stage breast cancer have increased as a result of the improvement of more widespread physical results and guidelines. In spite of this, a certain number of patients continue to develop distant metastases, which ultimately result in death.^[55] In addition to being the most aggressive subtype of BC with the worst prognosis and not responding to molecular targeted or endocrine therapy, TNBC has specific molecular phenotypes.^{[56, 57]} A major challenge in treating triple-negative breast cancer is overcoming chemotherapy resistance and recurrence. The diagnosis and treatment of TNBC require further research.

Despite the fact that almost 90% of cancer-specific deaths are caused by metastasis, the process of angiogenesis remains one of the most enigmatic aspects of the disease.^[56] Angiogenesis is a basic process for the growth and metastasis of malignant tumors.^[58] As tumors develop, oxygen and nutrients provided by blood are indispensable.^[59] However, despite considerable effort in developing anti-angiogenic drugs, these drugs are less effective and the disease eventually progresses.^[60] The concept of VM, which describes the ability of invasive cancer cells to produce fluid-conducting vascular-like structures without the involvement of endothelial cells, was discovered in 1999 in uveal melanoma and has attracted increasing interest from the scientific community.^{[61, 62]} Invasive disease, tumor spread, metastasis, and poor prognosis are associated with VM in patients.^{[63, 64]} As of now, there is no clear relationship between VM and angiogenesis. The results of several studies have shown that anti-angiogenic therapy does not alter VM, and that VM may also contribute to the maintenance of tumor growth during anti-angiogenic therapy.^{[62, 65]} According to existing scholarship, VM formation not only accelerates tumor metastasis, but also increases resistance to anti-angiogenic therapy. Additionally, anti-angiogenic therapy may result in the formation of VM, which allows drug-induced resistance to occur.^{[66, 67]} TNBC prognosis may be improved by using VM as a biomarker and potential therapeutic target. A better understanding of the complexity of VM may assist in the development of more effective and personalized antiangiogenic therapies for TNBC.

During the past decade, research has focused on the role of miRNAs in human cancers. Several miRNAs are associated with BC progression and overall survival, including, including miR-200b^[68], miR-124^[69], and miR-211^[70]. These miRNAs have shown promise as new targets for BC and other cancers.^{[71, 72]} Nevertheless, further research is needed on the majority of miRNAs in TNBC. It has been reported that miR-651-3p was dysregulated in various tumors and regulated the progression of glioma cancer^[17], ovarian cancer^[73], and hepatocellular carcinoma^[74] in the present work. In spite of this, miR-651-3p has not been thoroughly investigated in relation to TNBC. To improve the survival of patients with TNBC, it will be of great importance to investigate the molecular mechanisms by which miR-651-3p modulates the progression and the VM of the disease. To unveil the role of miR-651-3p in TNBC progression, we sought to identify the underlined mechanism. The present study, for the first time, provided clear evidence that miR-651-3p was found to be markedly up-regulated in TNBC tissues compared with normal tissues. Compared to the control group, miR-651-3p overexpression could significantly facilitate malignant biological behavior in TNBC cells. In a novel approach, we examined whether miR-651-3p expression level in TNBC also significantly influenced VM formation. Collectively, we concluded that miR-651-3p could be used as a potential therapeutic target due to its carcinogenic properties.

A number of studies have indicated that abnormal CAV1 expression or function is associated with tumor suppression and anti-proliferation.^{[75, 76]} In spite of this, the role of CAV1 in cancer remains unclear and controversial.^[77] In recent studies, it has been suggested that the expression level, type, and stage of the cancer, or the physical forces in the tumor environment may determine whether CAV1 acts as an oncogene or tumor suppressor.^[78] Chen^[79] reported that overexpression of CAV1 in HNSCC inhibited the process of ferroptosis, causing to aggressive phenotypes, as well as a worse prognosis. In BC, loss of CAV1 was associated with poor clinical outcomes.^[80] Nevertheless, conflicting results have shown that ectopic ATP5B promotes the migration and invasion of MDA-MB-231 cells by binding to CAV1.^[81] Due to these reasons, we investigated the role of CAV1 in TNBC progression and VM in our study. Our research has demonstrated that CAV1, as a downstream target of miR-651-3p, is essential for miR-651-3p-mediated promotion of TNBC malignancy behavior and VM formation. It was observed that the expression of CAV1 was significantly lower in TNBC tissues compared to paired normal tissues. Moreover, we found that downregulation of CAV1 inhibited proliferation, migration, and VM in TNBC by modulating the EGFR pathway, which is in line with previous studies.^{[33, 82]}

EGFR, receptor tyrosine kinases of the ErbB family, is a common molecular during carcinogenesis and an indispensable therapeutic target for the treatment of cancers, such as lung cancer, bladder cancer^[83], and colorectal cancer^[84]. EGFR promotes cancer proliferation, angiogenesis, and metastasis by binding to its ligand and activating downstream MAPK, AKT, and STAT3 signaling.^[85–88] Foxq1 regulates the EGFR signaling pathway, which may lead to VM in the nasopharyngeal.^[59] EGFR has not been studied in relation to the formation of VMs in TNBC. In a functional analysis of CAV1 PPI, we found that CAV1 may be involved in the activation of PI3K/Akt, MAPK pathways downstream of EGFR, which is consistent with previous studies.^[89] By inhibiting CAV1 transcriptional synthesis, miR-651-3p can enhance the activity of EGFR pathways, leading to promotion of TNBC proliferation, VM formation, and migration.

To summarize, this study explored for the first time the oncogene miR-651-3p, which is overexpressed in TNBC and is associated with proliferation, VM formation, and migration. Moreover, high levels of miR-651-3p in tumors result in a weakening of CAV1, thereby promoting TNBC progression by activating EGFR pathways. A possible therapeutic target for TNBC is the miR-651-3p/CAV1/EGFR axis, which has been identified in this study.

BC: Breast cancer; TNBC: Triple-negative breast cancer; ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2; VM: vasculogenic mimicry; miRNAs: Micro RNAs; NSCLC: non-small cell lung cancer; CAV1: Caveolin-1; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; KD: knockdown; OE: overexpression; NC: negative control; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; PBS: phosphate-buffered saline; BSA: bovine serum albumin.

Acknowledgments

The authors thank the staff of the Tianjin Medical University Cancer Institute & Hospital for providing helpful insights and suggestions.

Author Contributions

All authors participated in preparing the manuscript. Linyue Hai performed most of the experiments and wrote the manuscript. Jingjing Zhao performed some of the experiments. Xuchen Cao and Chunhua Xiao designed the experiments， revised critically for important intellectual content; and edited the manuscript. Chunhua Xiao is the guarantor of this work and has full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Data Availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Funding

This study was funded by Tianjin Key Medical Discipline (Specialty) Construction Project（TJYXZDXK-009A）and Wu Jieping Medical Foundation (320.6750.2021-10-2).

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No competing interests reported.

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MiR-651-3p/CAV1/EGFR axis Modulates Progression and Vasculogenic Mimicry in Triple-negative Breast Cancer

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Abstract

Figures

Introduction

Materials And Methods

Bioinformatics Analysis

Human Tissue

Cell Culture

Cell transfection

Immunofluorescence

RNA Extraction and Quantitative Real-Time PCR (RT-qPCR)

Dual-Luciferase Reporter Assay

Cell Proliferation Assay

Colony Formation Assay

Cell Migration Assay

Cell Cycle Analysis

Vasculogenic Mimicry Formation Assays

Western Blot Analysis

Statistical analysis

Results

miR-651-3p is dysregulated in TNBC tissues and cell lines

miR-651-3p regulates cell proliferation, migration, and VM formation of TNBC

CAV1 served as a direct target gene of miR-651-3p

CAV1 is aberrantly downregulated in pan-cancer tissues and correlates with disease progression

CAV1 Regulates TNBC cell proliferation, migration, and VM formation

MiR-651-3p regulates the EGFR signaling pathways by targeting CAV1

CAV1 overexpression resisted miR-651-3p-induced enhancement of TNBC proliferation, migration, and VM

Discussion

Abbreviations

Declarations

References

Additional Declarations

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