MicroRNA-335 inhibits tumor reinitiation and is silenced through genetic and epigenetic mechanisms in human breast cancer

Abstract

Post-transcriptional regulators have emerged as robust effectors of metastasis and display deregulated expression through unknown mechanisms. Here, we reveal that the human microRNA-335 locus undergoes genetic deletion and epigenetic promoter hypermethylation in every metastatic derivative obtained from independent patients' malignant cell populations. Genetic deletion of miR-335 is a common event in human breast cancer, is enriched for in breast cancer metastases, and also correlates with ovarian cancer recurrence. We furthermore identify miR-335 as a robust inhibitor of tumor reinitiation. We thus implicate the miR-335 locus on 7q32.2 as the first selective metastasis suppressor and tumor initiation suppressor locus in human breast cancer.

Figure 1.

Genomic copy number analysis reveals deletion of the miR-335 metastasis suppressor locus in multiple, independently derived metastatic cell derivatives. (A) Quantitative genomic real-time PCR was performed on DNA from the parental MDA-231 poorly metastatic breast cancer cell line as well as its highly lung metastatic (LM2) and bone metastatic (BoM2) derivatives using independent sets of primers (miR-335_a [orange], miR-335_b [yellow], and let-7e [green]). P-values represent unpaired one-tailed t-test significance values for differences between normalized copy number values of parental lines and each derivative line (n = 3). (*) *P < 0.005. All error bars represent SEM. (B) Quantitative genomic real-time PCR was performed on DNA from the CN34 primary malignant cancer population as well as its highly lung metastatic (LM1 and LM2), bone metastatic (BoM1), and brain metastatic (BrM2) derivatives. P-values represent unpaired one-tailed t-test significance values for differences between normalized copy number values between parental lines and each derivative line (n = 3). (*) P < 0.5; (**) P < 0.005. (C) The array-CGH plot reveals gross loss of copy number of chromosomal region on 7q encompassing miR-335 locus at 7q32.2 in MDA-231 metastatic derivatives. The plot depicts the log₂ ratio of array-CGH signals from various MDA-231 derivative lines relative to the array-CGH signal from reference normal genomic DNA. The parental MDA-MB-231 line is represented in black as biological replicates. Various metastatic derivatives are represented in color: 4142 (green), 4173 (orange), 4175 (light blue), 4180 (dark blue), and 831 (red). (D) The array-CGH plot depicts gross loss of copy number of chromosomal region at miR-335 locus at 7q32.2 in primary malignant CN34 metastatic derivatives. The parental CN34 line is shown in black, while its various metastatic derivatives are depicted in distinct colors.

Figure 2.

miR-335 is coregulated with the Mest transcript, and the miR-335/Mest promoter undergoes promoter hypermethylation in breast cancer. (A) The schematic of the Mest/miR-335 transcript reveals the location of miR-335 in the second intron of Mest. (B) Quantitative relationship of mature miR-335 levels to Mest transcript levels in breast cancer cell lines (correlation coefficient r² = 0.94; P < 0.0001). (C) Quantification of CpG density reveals three CpG islands in the miR-335/Mest promoter. (D) MSP of three CpG islands in Mest/miR-335 promoter from bisulfite-treated DNA of various lines. (NL. Genomic) Normal genomic DNA; (IVD) in vitro methylated DNA.

Figure 3.

Pyrosequencing reveals the methylation status of CpG island 3 as a predictor of miR-335 expression, and inhibition of DNA methylation is sufficient to restore miR-335 expression in metastatic breast cancer cells. (A–D) Pyrosequencing of bisulfite-treated DNA reveals the methylation percentage (Y-axis) as a function of CpG dinucleotide position in island 3 of poorly metastatic breast cancer populations (MDA parental and CN34 parental) and their highly metastatic derivatives (LM2 and CN34LM1A). All error bars represent SEM. (E) CpG methylation percentage as a function of miR-335 expression (correlation coefficient r² = −0.81; P = 0.004). (F) Quantitative real-time PCR expression of miR-335 expression in parental MDA-231 breast cancer line and its metastatic LM2 derivative and the CN34 primary malignant population and its metastatic derivative (CNLM1A) in the presence or absence of 5-Aza (5 μM treatment for 96 h for MDA lines and 1 μM for CN34 lines; metastatic lines, n = 6; parental lines, n = 3). P-values represent unpaired one-tailed t-test significance values. (*) P < 0.05; (**) P < 0.005. (G) Quantitative real-time PCR expression of Mest expression in parental MDA-231 breast cancer line and metastatic LM2 derivative in the presence or absence of 5-Azacytidine as in F; (n = 3). (*) P < 0.05; (**) P < 0.005.

Figure 4.

miR-335 suppresses breast cancer tumor initiation. (A) LM2 breast cancer cells (5 × 10⁵) expressing a control hairpin or miR-335 were implanted into the mammary fat pads of NOD–SCID mice. Events represent mammary injections at onset (green) and tumor palpation at 2 wk after implantation by two independent observers (red). LM2 breast cancer cells expressing a control short hairpin (1 × 10⁴ cells) (B) or miR-335 (1000 cells) (C) were implanted into the mammary fat pads of NOD–SCID mice. P < 0.0003 for statistical significance of difference between control (1000) and low-cell-number (10,000) cohorts. P < 0.006 for significance of difference between control and 1000 cohort; P-values represent one-tailed Fisher's exact test values. Bioluminescence images of mice 2 wk after implantation of LM2 Control-hairpin expressing (D) or miR-335 expressing (E) cells.

Figure 5.

The miR-335 locus is genetically inactivated in human breast cancer and during cancer progression as a result of focal and gross deletions. (A) Schematic representation of human chromosome 7 as well as a zoom schematic view of 7q32.2, the location of the human miR-335 locus. Red lines depict incidents of focal deletions in individual breast cancer tumors, as defined by one arm of the deletion falling within 2 Mb of the miR-335 locus. Arrows represent deletions spreading beyond the local region depicted. The inset summarizes frequencies of deletions (focal and gross) encompassing the miR-335 locus. The top percentage represents total incidence of deletions in the breast cancers of n = 353 patients, while the bottom percentage represents the incidence of deletions in the subset that developed metastatic relapse. (B) Quantitative real-time PCR of miR-335 expression in primary tumors and their respective metastases. (C) Quantitative genomic real-time PCR was performed on DNA from matched primary tumors and metastasis samples. (D) Kaplan-Meier curves for the ovarian cancer TCGA cohort depicting recurrence-free survival of patients whose tumors displayed deletion of miR-335 (blue) and those who did not (red). n = 228; P-value based on the log-rank test.