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. 2017 Mar 15;31(6):553-566.
doi: 10.1101/gad.292318.116.

miR-424(322)/503 is a breast cancer tumor suppressor whose loss promotes resistance to chemotherapy

Ruth Rodriguez-Barrueco  1   2 , Erin A Nekritz  1 , François Bertucci  3 , Jiyang Yu  4 , Felix Sanchez-Garcia  5 , Tizita Z Zeleke  1 , Andrej Gorbatenko  1 , Daniel Birnbaum  3 , Elena Ezhkova  6 , Carlos Cordon-Cardo  1 , Pascal Finetti  3 , David Llobet-Navas  1   2 , Jose M Silva  1
Affiliations

miR-424(322)/503 is a breast cancer tumor suppressor whose loss promotes resistance to chemotherapy

Ruth Rodriguez-Barrueco et al. Genes Dev. .

Abstract

The female mammary gland is a very dynamic organ that undergoes continuous tissue remodeling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy (in this case transiently) increase the risk of breast cancer, the reasons are unclear. Growing clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy. Here, through the analysis of ∼3000 primary tumors, we show that miR-424(322)/503 is commonly lost in a subset of aggressive breast cancers and describe the genetic aberrations that inactivate its expression. Furthermore, through the use of a knockout mouse model, we demonstrate for the first time that loss of miR-424(322)/503 promotes breast tumorigenesis in vivo. Remarkably, we found that loss of miR-424(322)/503 promotes chemoresistance due to the up-regulation of two of its targets: BCL-2 and insulin-like growth factor-1 receptor (IGF1R). Importantly, targeted therapies blocking the aberrant activity of these targets restore sensitivity to chemotherapy. Overall, our studies reveal miR-424(322)/503 as a tumor suppressor in breast cancer and provide a link between mammary epithelial involution, tumorigenesis, and the phenomenon of chemoresistance.

Keywords: breast cancer; chemoresistance; microRNA; tumor suppressor.

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Figures

Figure 1.
Figure 1.
miR-424(322)/503 is lost in breast cancer and is associated with poor prognosis and aggressive subtypes. (A) Copy number profile of the X chromosome in human breast cancers (METABRIC plus TCGA data sets), highlighting the chromosomal region containing the miR-424(322)/503 locus. The correlation between copy number and expression for surrounding genes is included at the bottom. The bar graph and box plots show the number of breast cancers presenting deletion (DEL) of the miR-424(322)/503 locus as well as the association between copy number and expression for miR-424(322) and miR-503. The bar graphs show the association between deletion of the miR-424(322)/503 locus with grade (B) and PAM50 molecular subtypes (C). (D) Overall survival of breast cancer patients with and without deletion of the miR-424(322)/503 locus (all patients and basal tumors are shown). (E) The miR-424(322)/503 cluster is monoallelically expressed as shown by sequencing a single-nucleotide polymorphism (SNP) located between the miRNAs in the cluster at the DNA and RNA level. Chromatin immunoprecipitation (ChIP) assays confirm that the expressed allele is associated with the active transcription elongation histone mark H3K36me3. (F) Methylation status analysis of the miR-424(322)/503 locus in breast cancers (TCGA-BRCA data set) with and without deletion of the locus. Each dot represents an individual primary tumor.
Figure 2.
Figure 2.
miR-424(322)/503−/− female mice develop mammary tumors that are promoted by pregnancy. (A) Carmine red staining of mammary glands from >1-yr-old miR-424(322)/503−/−, miR-424(322)/503+/−, and miR-424(322)/503+/+ virgins. (B) H&E and Ki-67 immunostaining of the samples from A. (C) Carmine red staining of mammary glands from >1-yr-old miR-424(322)/503−/− and miR-424(322)/503+/+ parous females. (D) H&E and Ki-67 immunostaining of the samples from C. (E) Quantification of the premalignant and malignant mammary lesions shown in B and D. χ2 tests and t-tests (Ki-67) were used to calculate P-values in E; P < 0.05 was considered significant. All mice were processed during the estrous phase (n ≥ 7). Parous animals had one pregnancy.
Figure 3.
Figure 3.
Primary miR-424(322)/503-deficient mammary tumors show aberrant up-regulation of targets CDC25A, BCL-2, and IGF1R. (A) RPPA data, available from the TCGA breast cancer database, were studied to correlate protein expression of BCL-2 and IGF1R with expression of miR-424. Two graphs are shown, comparing the 20% of samples presenting the highest miR-424 expression with the lowest 20% (left) and all samples (right). Red lines have been included as references. (B) Representative Western blots showing the expression levels of miR-424(322)/503 targets (IGF1R, BCL-2, and CDC25A) in the MMTV-Neu and MMTV-Neu-miR_KO models. The box plots show quantification of these data. The Western blots also show elevated phosphorylation (activation) of IGF1R and AKT. (C,D) Growth curves (C) as well as H&E and Ki-67 immunostaining (D) comparing tumors from MMTV-Neu and MMTV-Neu-miR_KO models. t-tests were used to calculate P-values; P < 0.05 was considered significant. For the animal studies, five to 10 animals were used per cohort.
Figure 4.
Figure 4.
Loss of miR-424(322)/503 induces resistance to anti-cancer therapy due to up-regulation of BCL-2 and IGF1R. (A) Mammary tumor volumes emerging in MMTV-Neu and MMTV-Neu-miR_KO animals were monitored until they reached ∼500 mm3, and then animals were treated twice per week with paclitaxel (PTX). (B) Growth curves showing the response to paclitaxel treatment. (C) Cleaved Caspase-3 immunostaining in tumors from MMTV-Neu and MMTV-Neu-miR_KO models after treatment with paclitaxel. (D) Illustration of the strategy to evaluate the use of ABT-199 and BMS-754807 to reverse chemoresistance in MMTV-Neu-miR_KO animals. (E) Growth curves showing the response to paclitaxel alone versus paclitaxel plus BMS-754807 and ABT-199. (F) Cleaved Caspase-3 immunostaining in the MMTV-Neu-miR_KO tumors from E. For animal studies, five to eight animals were used per cohort.
Figure 5.
Figure 5.
The impact of miR-424(322)/503 loss on the overall survival of breast cancer patients depends on chemotherapy treatment. (A) Overall survival of breast cancer patients (METABRIC and TCGA data sets) with and without deletion of the miR-424(322)/503 locus and with (+chemo) and without (−chemo) delivery of adjuvant chemotherapy. (B) Similar to A, but limited to patients with basal tumors. The statistical studies shown represent univariate analysis. Values for both univariate and multivariate analysis are shown in Table 1.
Figure 6.
Figure 6.
Model of transformation and induction of chemoresistance mediated by loss of miR-424(322)/503. (A) The miRNA cluster miR-424(322)/503 modulates the expression of multiples genes (including well-known oncogenes) that regulate various processes associated with tumorigenesis such as cell cycle progression, angiogenesis, immune response, cell metabolism, and cell adhesion. Note that a role for miR-424(322) and miR-503 in angiogenesis and immune evasion has been reported but was not evaluated in this study. Cells deficient in miR-424(322)/503 present aberrant high levels of these targets, leading to tumorigenesis. As some of these targets are controlled by the miR-424(322)/503 cluster during regression of the post-partum mammary epithelium, we propose that miR-424(322)/503-deficient cells are especially sensitive to transformation during pregnancy. (B) Loss of miR-424(322)/503 induces up-regulation of BCL-2 and IGF1R, which in turn promotes chemoresistance. Specific inhibitors against these proteins resensitized cancer cells to chemotherapy, representing a targeted therapeutic opportunity.
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