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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 2  |  Page : 345-350

Upregulation of CXC chemokine receptor 4-CXC chemokine ligand 12 axis ininvasive breast carcinoma: A potent biomarker predicting lymph node metastasis


Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Date of Web Publication8-Mar-2018

Correspondence Address:
Sadegh Babashah
Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-154, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.177221

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 > Abstract 


Objective: Breast cancer is considered as a heterogeneous disease, characterized by different biological and phenotypic features which make its diagnosis and treatment challenging. The CXC chemokine receptor 4 (CXCR4) expression was found to be correlated with poor overall survival in invasive breast carcinoma patients. Here, we sought to investigate the expression levels of the CXCR4-CXC chemokine ligand 12 (CXCL12) chemokine axis and their association with clinicopathologic features and lymph node metastasis in invasive breast carcinoma.
Materials and Methods: The expression of the CXCR4-CXCL12 chemokine axis and metastasis-related genes (E-cadherin and matrix metalloproteinase 2 [MMP2]) was measured by quantitative real-time-polymerase chain reaction. The correlation with various clinicopathologic parameters was analyzed.
Results: We found upregulation of the CXCR4-CXCL12 chemokine axis in invasive breast carcinoma samples compared with normal adjacent tissues. Moreover, we observed that upregulation of this chemokine axis was correlated with tumor stages and lymph node metastasis of breast tumors. Interestingly, this correlation was affected by the expression of human epidermal growth factor receptor 2/neu. There is also a significant correlation between the expression levels of CXCR4-CXCL12 axis and metastasis-related genes (E-cadherin and MMP2) in tumor samples with advanced stages of metastasis.
Conclusion: These results suggest a key role for the CXCR4-CXCL12 chemokine axis in breast cancer progression and highlight the prognostic importance of this chemokine axis for breast cancer survival.

Keywords: CXC chemokine receptor 4-CXC chemokine ligand 12 chemokine axis, invasive breast carcinoma, lymph node metastasis, prognosis


How to cite this article:
Dayer R, Babashah S, Jamshidi S, Sadeghizadeh M. Upregulation of CXC chemokine receptor 4-CXC chemokine ligand 12 axis ininvasive breast carcinoma: A potent biomarker predicting lymph node metastasis. J Can Res Ther 2018;14:345-50

How to cite this URL:
Dayer R, Babashah S, Jamshidi S, Sadeghizadeh M. Upregulation of CXC chemokine receptor 4-CXC chemokine ligand 12 axis ininvasive breast carcinoma: A potent biomarker predicting lymph node metastasis. J Can Res Ther [serial online] 2018 [cited 2019 Nov 22];14:345-50. Available from: http://www.cancerjournal.net/text.asp?2018/14/2/345/177221




 > Introduction Top


Breast cancer is the most frequently diagnosed cancer in women worldwide and a leading cause of cancer mortality. According to the National Cancer Institute, estimated new cases of breast cancer were about 233,000 women and about 40,000 people died from this neoplasm in the United States in 2014 (http://www.cancer.gov/).[1] There are currently several clinical and pathological factors which have shown prognostic importance for breast cancer survival. These include patient age, tumor size, menopausal status, lymph node involvement, hormone receptor status, human epidermal growth factor receptor 2 (HER-2) overexpression, and histological features (such as tumor grade and peritumoral vascular invasion).[2],[3] Although the determination of these prognostic algorithms for cancer risk stratification has led to improve the survival rate, one of the major challenges is that this approach does not consider the individual molecular complexity of each neoplasm. Indeed, accurate prediction of the metastatic potential of a tumor is critical in the management of breast cancer patients.[4]

The CXC chemokine receptor 4 (CXCR4) and its ligand 12 (CXCL12) (stromal cell-derived factor-1) have been shown to play a key role in migration, invasion, and adhesion of breast cancer cells, which promote tumor growth and metastasis. It is thought that the expression of CXCR4 on malignant epithelium cells in breast cancer results in directing the metastasis of CXCR4+ cancer cells to organs expressing high levels of CXCL12 (e.g., the regional lymph nodes, lung, liver, or bones).[5],[6],[7]

Since CXCR4-CXCL12 expression level is significantly correlated with breast cancer metastasis, we aimed to investigate the correlation of CXCR4-CXCL12 axis with lymph node metastasis. We also sought to study the important contributing factors such as HER-2 activation status. It seems that the CXCR4-CXCL12 chemokine axis may serve as a potent biomarker predicting lymph node metastasis in breast cancer. This highlights the prognostic importance of this chemokine axis for breast cancer survival.


 > Materials and Methods Top


Patients

Thirty-six pairs of specimens, tumor tissues and their adjacent normal ones, from patients with invasive breast carcinoma, were obtained from Iran's National Tumor Bank (Tehran, Iran). A record of clinical-pathological parameters including grading and staging (including tumor size, lymph node status, hormonal receptor status, and HER-2 status) for these tissue samples are summarized in [Table 1]. The study was approved by the Ethics Committee and Institutional Review Board, and informed consent was obtained from all the patients prior to the surgery. The specimens for assay were snap-frozen in liquid nitrogen and stored in −80°C until analysis.
Table 1: The clinical and pathological characteristics at diagnosis in invasive breast cancer patients

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RNA extraction and cDNA synthesis

Total RNA was extracted from frozen breast tissues using Trizol (Invitrogen) and treated with RNase-free DNase (Fermentas, Vilnius, Lithuania). The quality of RNA was verified by gel electrophoresis, and the concentration of RNA was assessed by optical density at 260 nm. Complementary DNA (cDNA) was synthesized by reverse transcription of 3 μg total RNA using random hexamers and RevertAid™ Reverse Transcriptase (Fermentas, ON, Canada).

Quantitative real-time polymerase chain reaction

Quantitative real-time polymerase chain reaction (Q-RT-PCR), based on SYBR green I chemistry, was performed on the ABI Step One Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Thermal cycling was performed using the following cycling conditions: 95°C for 5 min as first denaturation step, followed by 40 cycles of denaturation at 95°C for 10 s, and annealing/extension at 60°C for 30 s. Each individual Q-RT-PCR was followed by a dissociation stage; at 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s to ensure the homogeneity of PCR products. The mean threshold cycle was obtained from triplicate amplifications and used to calculate the amount of transcripts. Glyceraldehyde-3-phosphate dehydrogenase gene was used as housekeeping gene to normalize the amount of transcripts in each sample. The relative expression of each gene was calculated using the 2−ΔΔCt formula.[8] The amplification efficiencies of target and housekeeping genes were assessed from the standard curve drawn by plotting the logarithmic input amount of cDNA samples versus the corresponding Ct values. The corresponding real-time PCR efficiency (E) was calculated according to the slope of the standard curve and the equation: E = (10(−1/Slope)).[8] The primer sequences used in Q-RT-PCR assays are listed in [Table 2].
Table 2: The oligonucleotide primers used in quantitative real-time-polymerase chain reaction assay

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Statistical analysis

Data were presented as mean ± standard deviation of at least three experiments and analyzed by Student's t-test. The P values < 0.05 were considered statistically significant.


 > Results Top


Amplification efficiencies and primer specificity

Standard curves for target and housekeeping genes were drawn over serially diluted cDNA samples. As shown in [Supplementary Figure 1]a, the amplification efficiencies of investigated transcripts were approximately equal with high linear correlation, indicating the validity of the assay for relative quantification.



In addition, specificity of each amplification reaction was assessed by melting curve analysis. Results showed that no primer-dimer or detectable nonspecific products were generated during the applied Q-RT-PCR amplification cycles [Supplementary Figure 1]b.

Expression analysis of CXC chemokine receptor 4-CXC chemokine ligand 12 axis in breast carcinoma samples and their adjacent normal ones

To evaluate the activation status of the CXCR4-CXCL12 axis, we used Q-RT-PCR to examine the expression levels of CXCR4 and its chemokine ligand CXCL12 in 20 pairs of resected specimens, tumor and matched adjacent nontumor tissue samples, from patients with breast carcinoma. As shown in [Figure 1], the mean normalized ratios for CXCR4 and CXCL12 transcript levels in breast carcinoma specimens were remarkably higher than ratios found in corresponding normal tissues.
Figure 1: The CXC chemokine receptor 4-CXC chemokine ligand 12 axis is activated in invasive breast carcinoma. Transcript levels for CXC chemokine receptor 4 and CXC chemokine ligand 12 in invasive breast carcinoma samples and corresponding normal pairs were measured by quantitative real-time-polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase as housekeeping genes. Column, mean of three different experiments; bars, standard deviation

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Expression analysis of CXC chemokine receptor 4-CXC chemokine ligand 12 axis in breast carcinoma tissues with different clinical-pathological parameters

In order to investigate the correlation between expression levels of CXCR4-CXCL12 axis and the clinical-pathological parameters (tumor size, tumor stage, tumor grade, lymphatic metastases, nodal status, and perineural invasion), the expression levels of CXCR4-CXCL12 axis between the different groups were analyzed. As shown in [Figure 2]a, high expression levels of the chemokine receptor CXCR4 were observed in tumors with metastasis to lymph nodes [Figure 2]a. In this regard, there was a significant difference between stages I–II and stages III–IV of breast carcinoma tissues [Figure 2]b.
Figure 2: Significant difference of transcript levels for CXC chemokine receptor 4 and CXC chemokine ligand 12 in invasive breast carcinoma with different clinical and pathological characteristics: (a) Lymph node metastasis, (b) stages of tumor. Transcription levels were measured by quantitative real-time-polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase as housekeeping genes. Column, mean of three different experiments; bars, standard deviation

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We also sought to investigate whether the correlation between CXCR4 and lymph node metastasis was affected by positive or negative estrogen and progesterone receptor status. In this regard, this correlation was not associated with the hormone receptor status (data not shown). In addition, we found that there are no apparent differences of gene expression between the different groups classified by others parameters (data not shown).

Expression analysis of CXC chemokine receptor 4 in human epidermal growth factor receptor 2 positive breast carcinoma

It was previously showed that upregulation of CXCR4 is essential for HER-2-mediated tumor metastasis.[9] As HER-2 is a well-known biomarker associated with increased metastatic potential in breast cancer,[10],[11],[12] we sought to investigate whether the correlation between CXCR4 and lymph node metastasis is affected by the positive or negative expression of HER-2/neu. As compared to HER-2 negative breast carcinoma, all breast tumor tissues in which HER-2 expression was positive demonstrated significant upregulation of CXCR-4 transcript [Figure 3]. These results highlight a significantly positive correlation between HER-2 and CXCR4 expression.
Figure 3: A significant positive correlation between the expression of CXC chemokine receptor 4 and activation status of human epidermal growth factor receptor 2. Transcription levels were measured by quantitative real-time-polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase as housekeeping genes. Column, mean of three different experiments; bars, standard deviation

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Correlation between the expression levels of CXC chemokine receptor 4-CXC chemokine ligand 12 axis and metastasis-related genes (matrix metalloproteinase 2 and E-cadherin)

As previous reports have revealed that CXCR4 is able to alter the ability of cancer cells to adhere to the epithelium and invade through extracellular matrix components, we analyzed the expression levels of E-cadherin and matrix metalloproteinase (MMP2) genes in breast carcinoma samples.

In this regard, a high expression of MMP2 was found in breast tumor tissues as compared to corresponding normal pairs. Interestingly, a high expression of MMP2 in breast tumor tissues was strongly correlated with the expression levels of CXCR4-CXCL12 axis. However, no association was found between the expression levels of MMP2 in tumor tissues and tumor size (data not shown). As expected, there is also a significant negative correlation between the expression levels of CXCR4-CXCL12 axis and E-cadherin in tumor samples with advanced stages of metastasis [Figure 4].
Figure 4: Significant correlation between the expression levels of metastasis-related genes (matrix metalloproteinase 2 and E-cadherin) and CXC chemokine receptor 4-CXC chemokine ligand 12 activation status in breast carcinoma tissues with different stages of metastasis. Transcription levels were measured by quantitative real-time-polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase as housekeeping genes. Column, mean of three different experiments; bars, standard deviation

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 > Discussion Top


Breast cancer is considered as a heterogeneous disease, characterized by different biological and phenotypic features, which makes its diagnosis and treatment challenging.[13] The involvement of chemokines and their respective receptors in tumor development has been demonstrated in breast carcinoma, where CXCR4 overexpression is recognized as a requirement for breast cancer cell proliferation and is particularly correlating with poor prognosis.[9] In the present study, we sought to investigate whether the CXCR4-CXCL12 chemokine axis as a novel biomarker associated with increased metastatic potential in breast carcinoma could predict the presence of breast cancer metastases in lymph node-positive tumors.

A number of studies have revealed a correlation between CXCR4 expression and distant metastasis in primary breast cancer patients.[14],[15],[16] Wu et al.[17] reported that expression levels of the CXCR4 and CXCR7 chemokine receptors in breast cancer tissues were significantly higher than that in adjacent normal tissues. They suggested that the CXCL12-CXCR4-CXCR7 chemokine axis may serve as a critical factor in lymph node metastasis. In this regard, another study proposed that circulating levels of CXCL12 may serve as a prognostic blood marker predictive of distant metastasis in breast cancer.[18] These data were in consistent with our observations suggesting that the expression of CXCR4-CXCL12 chemokine axis is correlated with the status of lymph node metastasis.

The overexpression of HER-2 is observed in ~30% of all breast cancers and is associated with a relatively poor prognosis.[19] A positive correlation between HER-2 and CXCR4 expression has been previously suggested for breast cancer metastasis. Li et al.[9] revealed that HER-2 enhances the expression and function of CXCR4 by inhibiting CXCR4 degradation. In this regard, we sought to explore a possible direct linkage of CXCR4 and HER-2. As compared to HER-2 negative breast carcinoma, all breast tumor tissues in which HER-2 expression was positive demonstrated significant upregulation of CXCR-4 transcript [Figure 3]. These results lead us to suggest that HER-2 signaling in breast cancer may cause increased levels of CXCR4, which is required for HER-2 mediated invasion.

Reduced expression of E-cadherin is often observed in aggressive cancers and the loss of which is highly associated with epithelial-to-mesenchymal transition (EMT).[20],[21],[22] Acquisition of the invasive phenotype has many similarities with EMT, including the loss of cell–cell adhesion and increase in cell mobility. During EMT, there is a switch from E-cadherin expression to N-cadherin expression (a mesenchymal cell marker), which alters cell-matrix adhesion.[21] As previous reports have demonstrated that signaling through CXCR4 alters the ability of cancer cells to adhere to endothelium and invade through extracellular matrix components,[23],[24] we examined the expression levels of E-cadherin (as an epithelial cell marker) and MMP2 (as an important extracellular matrix component) in breast carcinoma samples. As expected, a significant positive correlation was observed between the expression levels of CXCR4-CXCL12 axis and MMP2 in tumor samples with advanced stages of metastasis [Figure 4]. However, no association was found between the expression levels of MMP2 in tumor tissues and tumor size (data not shown). Importantly, a low expression of E-cadherin in breast tumor tissues with advanced stages of metastasis was associated with high expression levels of CXCR4-CXCL12 axis [Figure 4].

Furthermore, we found that patients with increased expression levels of CXCR4 had more extensive metastases to lymph nodes relative to tumors with reduced CXCR4 expression [Figure 2]a. This data was in consistent with previous reports suggesting that high levels of CXCR4 were associated with decreased overall survival of patients.[5],[9] However, the possible functional role of correlation between CXCR4 and lymph node metastasis in breast carcinoma needs to be further investigated.

Taken together, our study revealed a new layer of molecular complexity which should be considered in the management of patients with invasive breast carcinoma. Further characterization of the central role of CXCR4 in invasive breast carcinoma will provide more insight into new signaling pathways, which are involved in breast cancer progression. In this regard, it is thought that CXCR4-CXCL12 chemokine axis may be a predictor of poor prognosis; however, the clinical significance of this chemokine axis remains to be further clarified.

Acknowledgments

The authors appreciate the valuable contribution of the patients included in this study. This work was supported by a research grant from Tarbiat Modares University.

Financial support and sponsorship

Research grant from Tarbiat Modares University.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

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