MicroRNA-543-3p a potential chemotherapy marker regulates the messenger RNA expression of survivin in patients with advanced breast cancer

Introduction

Patients with locally advanced carcinoma of the breast usually have a bad outcome when they managed by surgery and or radiotherapy.^[1] Therefore, induction chemotherapy (ICT) was used.^[2] ICT helps to improve the survival and locoregional of breast carcinoma.^[3] ICT provides many benefits: Large infiltrative malignant lesions can efficiently being reduced and removed by lumpectomy. Decreasing the extent of the surgery results in better cosmetic outcomes and reducing the locoregional recurrence.^[4] The other advantage of ICT is the testing of tumor sensitivity to chemotherapy.^[5] The major drawbacks of ICT are: Pre-operative treatment causes a lack of data on the status of the axillary nodes before chemotherapy and lengthens the time to surgery.^[6] Many clinical trials were conducted using ICT with variable numbers of cycles before surgical interventions. The published clinical response to ICT ranges between 30 and 90% with a 10–35% complete clinical response.^{[1, 7-9]}

Apoptosis is the process by which the body eliminates the senescent or damaged cells. This process is regulated by proteins that oppose or enhance cell death. Many cytotoxic drugs destroy malignant cells by interference with apoptosis.^[10] The malignant transformation in the cell is promoted by abnormal inhibition of apoptosis.^[11] Survivin is a protein that prevents the apoptotic pathways dependent or independent on caspase described as apoptosis inhibitors. It is located on chromosome 17q25.^[12] Survivin levels are usually low in non-malignant cells, but it is usually increased in several malignancies such as prostatic, colon, pancreas, lung, and lymphoid malignancies.^[13] Survivin expression showed a poor prognosis in most malignancies studied.^[14]

MiRNAs are short non-coding molecules consists of 20–22 nucleotides which negatively control gene expression at the post-transcriptional level. By sequence-guided recognition, miRNAs bound to the 3’-UTR of the target mRNA to induce mRNA degradation or translation repression.^[15-18] Many miRNAs seem to be important regulators in breast cancer cells such as cell division, metastases, and invasion. miR-542-3p is a part in these miRNAs, distinguished by the regulation of essential tumor-related mechanisms inhibiting malignant transformation.^[19,20] However, a little is published concerning the role of miR-542-3p in breast carcinoma. miR-542-3p regulates survivin mRNA by binding to one of three putative binding sites on its 3’-UTR. Upregulation of miR-542-3p leads to the downregulation of survivin and arrest of the cell cycle at G1 and G2/M phases resulting in tumor growth suppression.^[21]

The current study aims to evaluate the role of survivin and miRNA-542-3p in breast carcinoma and to study their role as molecular biomarkers for the prediction of the response to ICT.

Materials and Methods

Results

Survivin and miR-542-3p expression and the clinical response to ICT

Analysis of the levels of survivin-mRNA and miR-542-3p in BC and response to treatment revealed a significant correlation between survivin-mRNA levels and response. In patients with CR, survivin-mRNA was decreased with a mean ± SD value of survivin of 6.7 ± 0.11 while in patients with no response, it was elevated 18.97 ± 1.18 (mean ± SD). This variation was significant (P < 0.0001) [Figure 2a]. On the contrary, miR-542-3p was elevated in the complete responder with 2.85 ± 0.14 mean and SD value versus 0.39 ± 0.04 mean and SD value in patients with no response (P < 0.0001) [Figure 2b].

Table 2: Assessment of clinical response to induction chemotherapy

Response	Tumor		Nodes		χ2	P
	No.	%	No.	%
CR	15	29.4	15	29.4	17.3	0.001
PR	30	58.8	30	58.8
SD	6	11.8	6	11.8
Total	51	100	51	100

CR: Complete response, PR: Partial response, SD: Stable disease

Table 3: Assessment of pathological and clinical response at the tumor site after surgery

Pathological response**	Clinical response**		Total
	CR	PR	No.	%
No residual disease*	5	2	7	13.7
Microscopic residual disease*	11	2	13	25.5
Macroscopic residual disease*	3	28	31	60.8
Total	19	32	51	100

*(χ² =18.353, P<0.001), **(χ² =5.776, P=0.056)

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Figure 1:

Survivin-mRNA and miR-542-3p levels in BC and NABT. (a) Survivin-mRNA levels in BC and ANBT (P < 0.0001), (b) miR-542-3p levels in BC and ANBT (P < 0.0001), (c) Comparison of levels of survivin-mRNA and miR-542-3p in BC and ANBT, (P < 0.0001), and (d) relationship between levels of survivin-mRNA and miR-542-3p in BC revealed strong correlation (Pearson correlation)

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Survivin-mRNA and clinicopathological features

In our series of patients, we noted that increased levels of survivin-mRNA expression were significantly linked to poor clinical and pathological features. Increased expression was correlated with advanced-stage tumors (<0.00001), with premenopausal patients (<0.00001), with infiltrative lobular carcinomas (ILC) (<0.00001), with poorly differentiated tumors (<0.00001), with estrogen and progesterone negative tumors (<0.00001), and with HER2 positive tumors (P = 0.000132) [Table 4].

When we analyzed whether survivin-mRNA expression was linked to a worse prognosis, we calculated a univariate regression analysis with the clinicopathological features. No significant correlation between survivin-mRNA levels and other parameters except HER2 positivity (P = 0.0252). However, in a multivariate study, progesterone receptors (P < 0.05) and undifferentiated tumors (P = 0.018) were strongly correlated with survivin-mRNA expression [Table 5].

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Figure 2:

Levels of survivin-mRNA and miR-542-3p and treatment response. (a) Patients with complete response (CR) have low levels of survivin-mRNA (P < 0.0001). (b) Patients with CR have high levels of miR-542-3p (P < 0.0001). PR: Partial response, NR: No response

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Discussion

ICT is a standard of care for women with locally advanced, non-metastatic breast cancer and who needs to preserve their breasts by tumor reduction and at the same time keen about reducing locoregional failure.^[26] Besides these considerable advantages, ICT offers a basic advantage for evaluating the effect of chemotherapy on the tumor during surgery.^[27]

ICT acts by reducing the size and infiltration of breast primary and eliminating the systemic spread of malignant cells.^[28] Unfortunately, eliminating the systemic spread, malignant cells were widely neglected by the most of the studies, although cancer metastases are the primary cause of death in breast cancer and precise determination of micrometastatic cells the circulation is feasible,^[29,30] with a clinical validity in different types of cancer.^[31,32]

In the present study, we recorded 88.2% of the patients had a response to chemotherapy and CR was noted in 29.4% of them. Asselain et al.,^[2] in their meta-analysis, reported a 28% complete clinical response and 41% partial response after neoadjuvant chemotherapy. Furthermore, Klein et al.^[33] observed a CR in 25.2% of his patients and a partial response in 61.2%. These results support our results.

In the ACOSOG Z1071 clinical trial, Haffty et al. reported that 40.4% of their patients preserved their breast after ICT, 72.2% of their patients still have microscopic residue at the primary tumor site and 27.8% had pCR.^[34] In the current study, after chemotherapy and surgery, pathological examination of surgical specimens recorded 13.7% of patients with pCR to chemotherapy and 25.5% with microscopic residual disease. Our study revealed that 60.7% of our patients preserved their breasts after ICT.

To improve the outcome of women with advanced breast carcinoma, we sought to identify molecular markers to predict the chemotherapy response and try tailoring the therapy according to the specific features of the individual tumor.

We studied the relationship between survivin-mRNA and miRNA-542-3p in breast cancer as they are not fully investigated in breast cancer. The major criticism about ICT is the delay of surgery which may affect the local control. Hence, we studied whether we can use survivin-mRNA and miRNA-542-3p as potential molecular markers to predict response to chemotherapy.

Survivin acts as a controller for mitosis and an inhibitor of apoptosis. miR-542-3p target survivin at 3′-UTR regions inhibiting cell proliferation by inducing arrest of the cell cycle at G1 and G2/M. This means that miR-542-3p is the key regulator of survivin.^[21]

miR-542-3p was confirmed as the main regulator of survivinmRNA in several malignant tumors,^{[19, 35-37]} we evaluated if survivin is affected by miR-542-3p in breast cancer. At first, we estimated the levels of survivin-mRNA and miR-542-3p in breast cancer and then studied their clinical relation to breast cancer. Our finding showed a negative impact of the downregulation of miR-542-3p on survivin-mRNA.

In the current study, we noted a negative relationship between miRNA-542-3p and survivin. Upregulation of miRNA-542-3p results in the down-regulation of survivin mRNA. In the ANBT, the levels of miRNA-542-3p were significantly elevated and survivin-mRNA was significantly reduced. The same observation was also noted in neuroblastoma, bladder cancer, astrocytoma, and colorectal cancer.^{[19, 35-37]}

In our included patients, we found a strong relationship between levels of survivin-mRNA, miR-542-3p, and response. In complete responders to treatment, survivin-mRNA levels were low compared with high levels of miR-542-3p. On the other side, patients with no response to chemotherapy, survivinmRNA levels were highly increased compared to miR-542-3p levels which were significantly reduced.

In our series of patients, increased levels of survivin-mRNA had a significant relationship with adverse clinical and pathological features such as premenopausal status, estrogen and progesterone receptors negative tumors, HER2 receptor-positive tumors, ILC pathology, poorly differentiated tumors, and advanced tumor stage. Meanwhile, miR-542-3p levels were highly elevated in patients with good clinical and pathological parameters. Our observations were also reported by Zhang et al. in bladder cancer, they found that increased levels of survivin-mRNA and low miRNA-542-3p levels were linked to an advanced tumor and high rate of local recurrence.^[37] Ye et al. also noted that the relationship between low levels of miR-542-3p and bad prognostic features such as lymphatic and vascular infiltration, systemic metastases, and advanced disease in colorectal cancer.^[38]

On the contrary to our results and the others, Takeyama et al. have shown in their study in colorectal cancer that patients with hepatic metastases had an elevated miR-542-3p more than those without metastases.^[38] Such variation may be attributed to differences in the patient’s pathological features or genetic factors in the studied patients.

Since the miRNA-542-3p-survivin signal axis is not fully studied, and their role in breast cancer requires further studies and additional ideas into its functional mechanisms in breast cancer can be useful.

Conclusions

ICT is a reliable option for the treatment of advanced breast carcinoma. The studied miRNA-542-3p and its target survivinmRNA correlate negatively with each other in both malignant and their nearby normal tissues. miRNA-542-3p and survivin can be used as possible molecular markers to predict the results of treatment with chemotherapy.