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Clinical and biological impact of miR-18a expression in breast cancer after neoadjuvant chemotherapy

Cellular Oncology volume 42, pages 627–644 (2019)Cite this article

Abstract

Purpose

The analysis of breast cancer residual tumors after neoadjuvant chemotherapy (nCT) may be useful for identifying new biomarkers. MicroRNAs are known to be involved in oncogenic pathways and treatment resistance of breast cancer. Our aim was to determine the role of miR-18a, a member of the miR-17-92a cluster, in breast cancer behavior and outcome after nCT.

Methods

Pre- and post-nCT tumor miR-18a expression was retrospectively assessed by qRT-PCR in 121 patients treated with nCT and was correlated with survival outcomes and with clinical and pathological characteristics. Breast cancer-derived MCF-7 and MDA-MB-231 cell lines were transfected with miR-18a and anti-miR-18a to evaluate the biological effects of this molecule. In addition, whole-transcriptome expression analysis was performed.

Results

High miR-18a expression in post-nCT residual tumors was found to be associated with a significantly worse overall survival [hazard ratio (HR): 2.80, 95% confidence interval (CI): 1.01–7.76] and a strong trend towards a poorer disease-free survival (HR: 2.44, 95% CI: 0.99–5.02) compared to low miR-18a expressing post-nCT residual tumors. Clinical and experimental data were found to be in conformity with the proliferative effects of miR-18a, which showed a significant correlation with Ki67 and MYBL2 expression, both in pre- and post-nCT tumors and in public databases. In vitro analysis of the role of miR-18a in breast cancer-derived cell lines showed that a high expression of miR-18a was associated with a low expression of the estrogen receptor (ER), a decreased sensitivity to tamoxifen and an enrichment in luminal B and endocrine resistance gene expression signatures.

Conclusions

From our data we conclude that post-nCT miR-18a expression in breast cancer serves as a negative prognostic marker, especially in luminal tumors. Clinical, in vitro and in silico data support the role of miR-18a in breast cancer cell proliferation and endocrine resistance and suggest its potential utility as a biomarker for additional adjuvant treatment in patients without a pathologic complete response to neoadjuvant therapy.

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Acknowledgments

This study was funded by Instituto de Salud Carlos III (Projects PI12/02877 and PI15/1499), including FEDER funding by the European Union, and by Fundación Salud 2000 (Ayudas Merck Serono de Investigación 2012). ENM was funded by the Ministerio de Educación, Cultura y Deporte (Spain) (FPU16/06537). We acknowledge technical assistance by Dr. María José López Andreo (SAI-UM), Dr. Fara Sáez-Belmonte (SAI-UM), Charo Martínez-Marín (BIOBANC-MUR-IMIB), Lorena Velázquez (CRH), Nuria García Barberá (CRH-IMIB), Dr. Irene Martínez-Martínez (CRH-IMIB) and Natalia Bohdan (CRH-IMIB). We also acknowledge data managing support by Jose Antonio López Oliva and Natalia Andúgar (Hospital G. Universitario Morales Meseguer, Murcia, Spain).

Author information

Affiliations

  1. Servicio de Hematología y Oncología Médica, Hospital General Universitario Morales Meseguer y Centro Regional de Hemodonación, IMIB-Arrixaca, Avda. Marqués de los Vélez, s/n, 30008, Murcia, Spain

    Ginés Luengo-Gil, Elena García-Martínez, Esther Navarro-Manzano, Enrique González-Billalabeitia, Elisa García-Garre, Vicente Vicente & Francisco Ayala de la Peña

  2. Universidad de Murcia, Murcia, Spain

    Ginés Luengo-Gil, Esther Navarro-Manzano, Vicente Vicente & Francisco Ayala de la Peña

  3. Universidad Católica San Antonio, Murcia, Spain

    Elena García-Martínez, Pablo Conesa-Zamora & Enrique González-Billalabeitia

  4. Servicio de Anatomía Patológica, Hospital General Universitario Morales Meseguer, Murcia, Spain

    Asunción Chaves-Benito

  5. Servicio de Anatomía Patológica, Hospital General Universitario Santa Lucía, Cartagena, Spain

    Pablo Conesa-Zamora

  6. BiobancMur-Nodo 3, IMIB-Arrixaca, Murcia, Spain

    Alberto Martínez-Carrasco

Authors
  1. Ginés Luengo-Gil
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  2. Elena García-Martínez
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  3. Asunción Chaves-Benito
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  4. Pablo Conesa-Zamora
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  5. Esther Navarro-Manzano
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  6. Enrique González-Billalabeitia
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  7. Elisa García-Garre
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  8. Alberto Martínez-Carrasco
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  9. Vicente Vicente
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  10. Francisco Ayala de la Peña
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Corresponding author

Correspondence to Francisco Ayala de la Peña.

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All authors declare that they have no conflict of interest.

Informed consent

All procedures performed in this study involving patients were in accordance with the ethical standards of the institutional research committee and with the Declaration of Helsinki. Written informed consent was obtained from all patients included in the study, and the study was approved by the hospital Ethics and Clinical Research Committee (CEIC Hospital G. Universitario Morales Meseguer; reference number: ESTU-23/12).

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Fig. S1
figure 9

Comparative analysis of miR-18a expression (before and after chemotherapy) depending on hormone receptors (HR) and HER2 status. (a) MiR-18a expression was significantly decreased in HR + HER2- tumors before and after treatment (pre-CT n: yes = 45, no = 50, p = 0.001; post-CT n: yes = 50, no = 46; p = 0.013). (b) There were no differences in miR-18a expression in HR + HER2+ tumors (pre-CT n: yes = 14, no = 57, p = 0.170; post-CT n: yes = 13, no = 56; p = 0.771). (c) There were no differences in miR-18a expression in HR-HER2+ tumors (pre-CT n: yes = 13, no = 82, p = 0.119; post-CT n: yes = 11, no = 85; p = 0.726). (JPG 1110 kb)

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Fig. S2
figure 10

MiR-18a expression among different breast cancer subtypes after chemotherapy. After treatment, significant differences were observed only between HR + HER2- and TNBC tumors (Kruskal-Wallis Test, p = 0.0443; HR + HER2- n = 50, HR + HER2+ n = 13, HR-HER2+ n = 11 and TNBC n = 22). (JPG 396 kb)

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Fig. S3
figure 11

Validation analysis of miR-18a expression by breast cancer subtype. (a) In our validation series (Cartagena) (Kruskal-Wallis Test, p = 0.019; luminal A, n = 18; luminal B, n = 42; HR + HER2+, n = 15; HR-HER2+, n = 8; TNBC, n = 14). (b) In Oslo external series (GSE19536) (Kruskal-Wallis Test, p = 0.003; luminal A, n = 41; luminal B, n = 12; HER2+, n = 17; basal, n = 15). (c) In TCGA external series (Kruskal-Wallis Test, p < 0.0001; luminal A, n = 342; luminal B, n = 152; HER2+, n = 55; basal, n = 109). (JPG 1386 kb)

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Fig. S4
figure 12

Baseline expression of miR-18a in different cell lines. MiR-18a expression in the luminal model, MCF7, was significantly lower than that in the triple negative model, MDA-MB-231 (p = 0.049); expression in the luminal model, MCF7, was significantly lower than that in the HER2+ model, SK-BR-3 (p = 0.049); the HER2+ model, SK-BR-3 did not significantly differ from the triple negative model, MDA-MB-231 (p = 0.513) (n = 3 per group, three independent experiments). (JPG 591 kb)

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Fig. S5
figure 13

Expression of miR-18a depending on the level of ki67 (IHC). (a) Using a cutoff point of 14%. (b) Using a cutoff point of 20%. In both cases, the expression of miR-18a was associated with increased expression of ki67. N values for each group are shown in the figure. (JPG 441 kb)

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Fig. S6
figure 14

Efficient miR-18a transfection by RT-qPCR. Prior to array experiments, efficient transfection of MCF7 cells under all conditions was assessed by measuring the expression levels of miR-18a. (JPG 199 kb)

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Fig. S7
figure 15

Absence of changes in EMT markers after transfection of MCF7 cells with miR-18a and anti-miR18a. (a) Vimentin mRNA expression (n = 3 per group, three independent experiments). (b) Cadherin-1 mRNA expression (n = 3 per group, three independent experiments). (JPG 535 kb)

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Fig. S8
figure 16

Lack of modification of angiogenic marker expression (western blot) after transfection of MCF7 with miR-18a. (a) MiR-18a does not affect the protein expression of thrombospondin-1. (b) MiR-18a does not affect the protein expression of platelet-derived growth factor subunit B. (c) MiR-18a does not affect the protein expression of hypoxia-inducible factor 1-alpha. (d) MiR-18a does not affect the protein expression of vascular endothelial growth factor A. (JPG 2241 kb)

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Fig. S9
figure 17

Analysis of the effects of miR-18a on MDA-MB-231 in vitro. (a) Overexpression of miR-18a showed a slight but significant increase in the proliferation index of MDA. (b) MiR-18a conditioned medium from MDA showed no angiogenic effects on the EA.hy926 endothelial cell model. (c) Impaired migration was observed after transfection with both miR-18a and anti-miR-18a. (d) MiR-18a did not affect the invasive features of MDA (JPG 16905 kb)

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Fig. S10
figure 18

Association between ESR1/ERα and miR-18a expression. Expression of the estrogen receptor (mRNA: a, c; protein by immunohistochemistry: b, d) was inversely correlated with miR-18a in both prechemotherapy (a, b) and postchemotherapy (c, d) biopsies. (JPG 483 kb)

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Luengo-Gil, G., García-Martínez, E., Chaves-Benito, A. et al. Clinical and biological impact of miR-18a expression in breast cancer after neoadjuvant chemotherapy. Cell Oncol. 42, 627–644 (2019). https://doi.org/10.1007/s13402-019-00450-2

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Keywords

  • Breast cancer
  • Neoadjuvant chemotherapy
  • Prognostic markers
  • miR-18a
  • miR-17-92a
  • Endocrine resistance