HnRNPA2/B1 Is a Novel Prognostic Biomarker for Breast Cancer Patients

Abstract

Aims:

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is highly expressed in multiple types of tumor tissues and could potentially be used as a biomarker for the early detection of lung cancer. However, there is little evidence supporting its clinical significance as a prognostic marker in breast cancer.

Materials and Methods:

We retrospectively analyzed the protein expression and localization of hnRNPA2/B1 protein in breast cancer tissues and adjacent normal tissues from 50 patients with Stage II and III breast cancer who were treated at Shanxi Provincial People's Hospital from May 2018 to May 2019 using western blot, and immunofluorescent and immunohistochemical staining assays. In addition, bioinformatic analyses using the Affymetrix Human Genome database were performed to examine the mRNA levels of hnRNPA2/B1 in normal and breast cancer tissues, and to determine their correlation with the survival rates of breast cancer patients.

Results:

Based on the cohort of 50 patients, HnRNPA2/B1 protein was expressed in both the cytoplasm and nucleus of breast cancer cells. The protein levels of hnRNPA2/B1 in breast cancer tissues were significantly higher than those in adjacent normal tissues (p < 0.001). Furthermore, bioinformatic analyses of hnRNPA2/B1 mRNA expression levels demonstrated that they were negatively correlated with overall survival and disease-specific survival rates in breast cancer patients.

Conclusion:

Our study indicates that hnRNPA2/B1 could serve as a novel prognostic biomarker for breast cancer.

Introduction

Heterogeneity nuclear proteins (hnRNP) represent a large family of RNA-binding proteins with a variety of functions, including DNA repair, telomere maintenance, transcription, packaging, and processing of newborn heterogeneous nuclear RNA (hnRNA), splicing of primary transcripts (pre-mRNAs), nuclear shuttle, and translational control. HnRNPA2/B1 is a crucial member of the hnRNP family. This protein is encoded by a single-copy gene located on human chromosome p15. HnRNPA2/B1 participates in the packaging and splicing of pre-mRNAs, nucleo-cytoplasm transport, and translational regulation of mRNA, thereby influencing various physiological and pathological processes (Chen et al., 2010; Geuens et al., 2016). The pre-mRNA of hnRNPA2/B1 is variably spliced into B1, A2 (which lacks exon 2 compared with B1), B1b (which lacks exon 9 compared with B1), and A2b (which lacks both exons 2 and 9 compared with B1) variants. Each variant is differentially expressed in different tissues, at different disease stages, and in individuals at different ages, suggesting the different functions of these hnRNPA2/B1 protein isoforms encoded by different mRNA variants (Meng et al., 2019). However, most functional studies on hnRNPA2/B1 have focused on the A2 isoform, which is highly expressed in various tissues. Thus, it is difficult to distinguish among these isoforms due to a lack of isoform-specific antibodies.

To date, only B1 and A2 isoforms have been found to be expressed in human cells (Geuens et al., 2016; Meng et al., 2019). HnRNPA2/B1 participates in a variety of physiological and pathological cell processes. As a self-antigen, high expression of hnRNPA2 is related to the pathogenesis of rheumatoid arthritis (Fritsch et al., 2002; Trembleau et al., 2010). HnRNPA2 plays a regulatory role in DNA replication, transcription, and recombination by binding to telomere DNA repeats and RNA molecules in telomerase to maintain telomere structure (Ford et al., 2002; Moran-Jones et al., 2005). HnRNPB1 affects the stability of the genome and regulates cell cycle and apoptosis by inhibiting the activity of DNA-dependent protein kinase (DNA-based protein kinase) (Han et al., 2008). HnRNPB1 can also participate in the SUMOylation regulation pathway and is related to various cell processes (Maggipinto et al., 2004; Moran-Jones et al., 2009).

HnRNPA2/B1 contains a nuclear localization signal (NLS) and is mainly detected in the nucleus under normal circumstances; however, alternative pre-mRNA splicing leads to the deletion of the NLS, which renders hnRNPA2/B1 cytoplasmic localization. For example, hnRNPA2b isomer was observed in the cytoplasm of mouse nerve cells (Meng et al., 2019). In addition, hnRNPA2 isomer detected in the cytoplasm of human nerve cells was proposed to interact with TOG (tumor overexpressed gene) proteins and tubulin (Brumwell et al., 2002; Kosturko et al., 2005).

Breast cancer is the most common type of cancer in women, accounting for approximately one-third of the total cancer cases in women and >10% of all types of cancers globally (Jemal et al., 2010). Prognostic biomarkers provide an effective tool for early detection of breast cancers, enabling a more benefit-risk-balanced and personalized treatment on the patients (Polley et al., 2013). For decades, multiple molecular markers, including breast cancer susceptibility gene 1 (BRCA1) (Santarosa et al., 2010), have been developed and deployed as routine prognostic markers to characterize the type of cancer and inform therapeutic approaches. As an important RNA-binding protein that affects RNA processing, splicing, transport, and stability of many genes, the function of hnRNPA2B1 in the occurrence and progression of breast cancer has not been clearly defined. In a previous study, the overexpression of hnRNPA2/B1 was observed in BRCA1-mutated breast and ovarian cancers, especially in BRCA1-deficient cells and tumors (Santarosa et al., 2010). In another study, the authors observed higher hnRNPA2B1 levels in breast cancer tissues than normal tissues (Zhou et al., 2001). It was also revealed that hnRNPA2/B1 silencing in breast cancer cells can inhibit their proliferation (Golan-Gerstl et al., 2011; Deng et al., 2016; Hu et al., 2017). Klinge et al. (2019) reported the upregulation of hnRNPA2/B1 in endocrine-resistant LCC9 breast cancer cells, which altered the microRNA transcriptome in MCF-7 cells. Liu et al. (2020) reported HNRNPA2/B1 as a negative regulator of human breast cancer metastasis by maintaining the balance of multiple genes and pathways. However, it is still unclear if hnRNPA2/B1 is a prognostic factor for breast cancer. Therefore, the objective of the present study was to characterize the clinical significance of hnRNPA2B1 as a prognostic biomarker in breast cancer.

Materials and Methods

Ethics approval and study subjects

This study was approved by the local Ethics Committee of Shanxi Provincial People's Hospital, China (No: 201910), and the requirement for informed patient consent was waived. All procedures were performed in strict compliance with the Declaration of Helsinki. We retrospectively obtained tumorous and normal tissues from 50 stage II or stage III breast cancer patients who were treated with surgery in Shanxi Provincial People's Hospital during May 2018 to May 2019. The selection of patient cohort was based on that these patients with stage II or stage III breast cancer only needed surgery treatment.

Inclusion criteria for this study were as follows: (1) patients who were diagnosed with stage II or stage III breast cancer based on histopathological examination results. (2) Patients who were treated only with surgery, but not with chemotherapy, radiotherapy, or other targeted drug therapy. (3) Patients who did not have other types of cancer. (4) Patients who did not have fatal complications. (5) Patients who did not have genetic mutations that affect breast cancer prognosis. We also excluded the patients with a long history of smoking or drinking, and those with familial genetic diseases.

Immunohistochemistry

Adjacent normal tissue and breast cancer sections (5 μm in thickness) were deparaffinized and rehydrated with a gradient of ethanol concentrations. Antigen retrieval was conducted in EDTA (PH 8.0 and heated in a microwave oven with the power set on “high” (100% power) for 3 min and on “low” (20% power) for 15 min. After antigen retrieval, the sections were continuously incubated in three solutions: (1) hnRNPA2/B1 antibody at 1:100 dilution (xmu-ssl-1B; Bam Biotech Co., Xiamen, China); (2) biotinylated secondary antibody (Boster, Wuhan, China) solution, and (3) avidin-conjugated horseradish peroxidase solution.

All images were acquired using a Nikon Trinocular microscope (Nikon Ni U, Japan) and analyzed using Image-Pro Plus software (Media Cybermetics, Rockville).

Immunofluorescence

The breast cancer and adjacent normal tissues were embedded in optimal cutting temperature compound and cut into 3-μm sections. The sections were set aside for 30 min at room temperature and fixed with acetone at room temperature for 5-10 min. After washing three times with phosphate-buffered saline for 10 min and microwave-antigen retrieval, the sections were blocked with goat serum blocking buffer (Boster) at room temperature for 60 min, and incubated with primary antibody (hnRNPA2/B1 1:100) at 4°C overnight. The sections were incubated with secondary antibody (sheep-anti-rabbit IgG Alexa Fluor 555 conjugate antibody; Life Science Technology, Carlsbad, CA) at room temperature for 1 h and counterstained with 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI) to label the nuclei (Boster). HnRNPA2/B1 staining was visualized using confocal immunofluorescence microscopy (Olympus FV-1000, Japan).

Western blot analysis

The breast cancer and adjacent normal tissues were homogenized in lysis buffer (Boster). Equal amounts of protein (50 μg), as determined by a bicinchoninic acid assay kit (Boster), were separated using 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). After blocking, the membranes were incubated overnight at 4°C with primary antibodies (hnRNPA2/B1 1:300; β-actin 1:10,000; Abcam). After washing three times, the membranes were incubated with the secondary antibody (1:5000; Boster) at room temperature for 2 h and the protein bands were visualized using the Quantity One analysis system (Bio-Rad, Hercules, CA).

Data analysis

The Illumina HiSeq RNA-seq results based on 112 breast cancer tissues and 1073 adjacent normal tissues deposited in the Cancer Genome Atlas (TCGA)_RNA_Seq database and the Affymetrix Human Genome U133A microarray data of a GSE5847_GPL96 cohort were used (PubMed ID:17999412, included 48 breast cancer tissues and 47 adjacent normal tissues) to comparatively analyze the mRNA levels of hnRNPA2/B1 in normal and breast cancer tissues.

The Affymetrix Human Genome U133A microarray data of a GSE1456_GPL96 cohort (which included 157 Swedish breast cancer patients with 26 and 131 patients in high and low HnRNPA2/B1 expression groups, respectively) and the Affymetrix Human Genome U133B microarray data of a GSE1456_GPL97 cohort (which includes 150 Swedish breast cancer patients with 11 and 139 patients in high and low HnRNPA2/B1 expression groups, respectively) were used to analyze the correlation between the mRNA level of hnRNPA2/B1 in breast cancer tissues and the overall survival (OS) or disease-specific survival (DS) of the patients.

HnRNPA2/B1 copy number in 1055 breast cancer patients was determined based on their Genome Wide Human SNP Array data deposited in the TCGA_CNV database.

Experimental data with three biological repeats were statistically analyzed with paired t-tests using SPSS 23.0 (IBM SPSS, Chicago, IL) and are expressed as mean ± standard error of the mean. The Kaplan-Meier estimator was used for OS and DS analyses, and the magnitude and significance of the difference between groups were measured by hazard ratio and p value, respectively. In this study, p < 0.05, p < 0.01, and p < 0.001 represent significant, very significant, and extremely significant differences, respectively.

Results

Protein expression levels and subcellular location of hnRNPA2/B1 in breast cancer and adjacent normal tissues

To comparatively analyze the protein levels of hnRNPA2/B1 in breast cancer and paired normal tissues, we performed immunohistochemical staining and western blot analyses on the tissue samples collected from the 50 enrolled breast cancer patients in the hospital. The results showed that the protein levels of hnRNPA2/B1 were significantly higher in breast cancer tissues compared with the adjacent normal tissues (Fig. 1A, C).

FIG. 1.

The protein levels of hnRNPA2/B1 in breast cancer and adjacent normal tissues. (A) Immunohistochemistry analysis (original magnification: × 400) of the protein levels of hnRNPA2/B1 in normal tissues (left panel) and breast cancer tissues (middle panel), and densitometric analysis (right panel). **p < 0.01. (B) Subcellular localization of hnRNPA2/B1 in breast cancer (lower panels) and adjacent normal tissues (upper panels), visualized using confocal microscopy (original magnification: × 400). (C) Western blot analysis of the protein levels of hnRNPA2/B1 in breast cancer and adjacent normal tissues. Left panel, western blotting results. Right panel, densitometric analysis. ****p < 0.0001. DAPI, 4′6′-diamidino-2-phenylindole dihydrochloride; hnRNPA2/B1, heterogeneous nuclear ribonucleoprotein A2/B1. Color images are available online.

We then determined the subcellular localization of hnRNPA2/B1 in the cells of breast cancer and normal tissues. HnRNPA2/B1 proteins in all the tissue samples were immunofluorescently labeled, and all nuclei were stained with DAPI. Expression and subcellular localization of hnRNPA2/B1 were visualized using laser scanning confocal microscopy. According to our analysis, being different from that in the adjacent normal tissues, the fluorescent signal of hnRNPA2/B1 can be detected in both the cytoplasm and the nuclei of cells in breast cancer tissues (Fig. 1B). These results indicate that hnRNPA2/B1 was expressed at high levels in nuclei and cytoplasm of breast cancer cells.

Transcript levels of hnRNPA2/B1 in breast cancer and adjacent normal tissues

To comparatively analyze the mRNA levels of hnRNPA2/B1 in breast cancer and paired normal tissues, we utilized two sets of published microarray data. As shown in Figure 2A, the mRNA levels of hnRNPA2/B1 were significantly higher in the 112 breast cancer tissues from TCGA than those in the 1073 adjacent normal breast tissues (p < 0.001). Compared with those in the 47 normal tissues in cohort GSE5847_GPL96, the mRNA levels of hnRNPA2/B1 were also significantly higher in the 48 breast cancer tissues in cohort GSE5847_GPL96 (p < 0.001) (Fig. 2B).

FIG. 2.

The mRNA levels of hnRNPA2/B1 in breast cancer and paired normal tissues. (A) hnRNPA2/B1 mRNA levels in the 112 breast cancer tissues and 1073 adjacent normal breast tissues from TCGA. (B) hnRNPA2/B1 mRNA levels in the 47 normal tissues and 48 breast cancer tissues in cohort GSE5847_GPL96. TCGA, The Cancer Genome Atlas. Color images are available online.

Correlations between the mRNA levels of hnRNPA2/B1 and survival rates of breast cancer patients

Given that the mRNA levels of hnRNPA2/B1 were significantly higher in breast cancer tissues than those in the adjacent normal tissues, we next investigated the potential correlation between hnRNPA2/B1 mRNA levels and survival rates of breast cancer patients. As shown in Figure 3, the mRNA level of hnRNPA2/B1 was significantly and negatively correlated with the survival rate of breast cancer patients. For the 157 Swedish breast cancer patients in cohort GSE1456_GPL96, the OS rate of the patients with high hnRNPA2/B1 expression was 56.6%, while that of the patients with low hnRNPA2/B1 expression 76.8% (Fig. 3A). The DS rate of the patients with high hnRNPA2/B1 expression was 58.8%, while that of the patients with low hnRNPA2/B1 expression group was 84.1% (Fig. 3B). These results indicate that the mRNA level of hnRNPA2/B1 was significantly and reversely correlated with both the OS and DS rates of breast cancer patients (p < 0.05). For the 150 Swedish breast cancer patients in cohort GSE1456_GPL 97, the OS rate of the patients with high hnRNPA2/B1 expression group was 54.5%, while that of the patients with low hnRNPA2/B1 expression group was 75.5% (Fig. 3C). The DS rate of the patients with high hnRNPA2/B1 expression group was 72.5%, while that of the patients with low hnRNPA2/B1 expression group was 84.0% (Fig. 3D). These results demonstrate that the mRNA level of hnRNPA2/B1 was significantly and reversely correlated with the OS rate in breast cancer patients (p < 0.05), while no significant correlation existed between the mRNA level of hnRNPA2/B1 and the DS rate of the patients (p > 0.05).

FIG. 3.

Correlations between hnRNPA2/B1 mRNA levels and the survival rates of breast cancer patients. (A) The correlation between hnRNPA2/B1 mRNA level and OS rate in 157 breast cancer patients included in cohort GSE1456_GPL 96. (B) The correlation between hnRNPA2/B1 mRNA level and DS rate in 157 breast cancer patients included in cohort GSE1456_GPL 96. (C) The correlation between hnRNPA2/B1 mRNA level and OS rate in 150 breast cancer patients included in cohort GSE1456_GPL 97. (D) The correlation between hnRNPA2/B1 mRNA level and DS rate in 150 breast cancer patients included in cohort GSE1456_GPL 97. DS, disease-specific survival; OS, overall survival. Color images are available online.

Variations in hnRNPA2/B1 gene copy number in breast cancer patients

Finally, we designated to explore the mechanism underlying the high expression of hnRNPA2/B1 in breast cancer tissues by analyzing hnRNPA2/B1 gene copy numbers in breast cancer patients. As shown in Figure 4, among the 1055 breast cancer patients included in the TCGA_CNV database, 31.4% had increased hnRNPA2/B1 gene copy numbers, while 10.4% had decreased hnRNPA2/B1 gene copy numbers.

FIG. 4.

Variations in hnRNPA2/B1 gene copy number in breast cancer patients included in the TCGA_CNV database. The blue bar represents the percentage of patients with decreased hnRNPA2/B1 gene copy numbers, while the red bar represents the percentage of patients with increased hnRNPA2/B1 gene copy numbers. Color images are available online.

Discussion

The present study reveals that hnRNPA2/B1 expression was higher in breast cancer tissues compared with that in normal tissues, and this increase in hnRNPA2/B1 expression was significantly associated with poor OS. Furthermore, increased copy numbers of hnRNPA2/B1 gene were observed in nearly one-third of breast cancer patients. Our findings suggest that hnRNPA2/B1 has a great potential in serving as a biomarker to distinguish breast cancer tissues from normal tissues. Thus, hnRNPA2/B1 could be a promising molecular target for the development of anti-breast cancer drugs.

In the present study, hnRNPA2/B1 proteins were predominantly expressed in cell nuclei, followed by the cytoplasm, which was consistent with the findings from previous studies based on various tumors (Kamma et al., 1999; Sueoka et al., 1999; Satoh et al., 2000; Kamma et al., 2001; Zhou et al., 2001; Yan-Sanders et al., 2002; Wu et al., 2003; He et al., 2005; Mizukami et al., 2005; Sueoka et al., 2005; Jing et al., 2011; Liang et al., 2011; Meng et al., 2019). The high expression and cytoplasmic distribution of hnRNPA2/B1 in a variety of cancer cells were previously demonstrated (Satoh et al., 2000; Zhou et al., 2001; Yan-Sanders et al., 2002; Jing et al., 2011; Liang et al., 2011), suggesting the important role of hnRNPA2/B1 isoforms in posttranscriptional regulation in both the nuclear and cytoplasmic compartments. HnRNPA2/B1 levels have been shown to be associated with the degree of cell differentiation and proliferation (Golan-Gerstl et al., 2011; Jing et al., 2011; Liang et al., 2011). These two isoforms also participate in the regulation of epithelial-mesenchymal transition in lung cancer epithelial cells (Boukakis et al., 2010; Tauler et al., 2010). Therefore, aberrant cytoplasmic expression of hnRNPA2/B1 is recognized as an early molecular event in lung cancer and serves as an early diagnostic biomarker (Fielding et al., 1999; Katsimpoula et al., 2009; Tauler et al., 2010).

So far, little is known about the function of hnRNPA2/B1 in breast cancer development and progression. In the present study, the differential expression of hnRNPA2/B1 in breast cancer and adjacent normal tissues demonstrated that hnRNPA2/B1 could serve as an early diagnostic biomarker for breast cancer. Furthermore, the negative correlations between hnRNPA2/B1 mRNA level and OS rate of breast cancer patients suggest an OS-predictive function of hnRNPA2/B1 in breast cancer patients. However, the correlation between hnRNPA2/B1 mRNA level and the DS rate was not always significant, possibly due to the less number of patients and larger difference in the patient numbers between groups in GSE1456_GPL97 cohort. More studies are needed to determine the expression patterns of different hnRNPA2/B1 isoforms and their isoform-specific functions in breast cancer.

We also observed the changes of hnRNPA2/B1 gene copy number in breast cancer tissues. Frequent alterations of several splicing factors, including hnRNPA2/B1, have been observed to change gene copy number and/or expression level in solid tumors (Anczukow and Krainer, 2016). HnRNPA2/B1 gene encodes 2 isoforms, among which the B1 isoform has 12 additional amino acids near the N-terminus. In the present study, hnRNPA2/B1 was highly expressed in breast cancer tissues, which could be partially attributed to the changes of hnRNPA2/B1 gene copy number. However, this speculation needs to be verified by future in vitro and in vivo studies.

Our present study has some limitations. First, the correlation between DS rate and hnRNPA2/B1 mRNA levels in the 150 Swedish breast cancer patients in cohort GSE1456_GPL 97 was not statistically significant. Second, all of our findings were based on retrospective analysis of TCGA datasets without clinical or biological validation. Future studies with a larger cohort are needed to confirm the correlation between hnRNPA2/B1 expression and OS in breast cancer patients.

Conclusion

In summary, hnRNPA2/B1 is highly expressed in breast cancer tissues compared with adjacent normal tissues. We also reveal a significantly negative correlation between hnRNPA2/B1 transcript level and OS in the patients with breast cancer. The findings of the present study demonstrate the potential of hnRNPA2B1 as a promising clinical prognostic biomarker for breast cancer.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was financially supported by the National Natural Science Foundation of China (grant number: 81570626) and the National Science Foundation of Shanxi Province of China (grant number: 201701D111001).

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