Genomic DNA copy-number alterations of the let-7 family in human cancers

Abstract

In human cancer, expression of the let-7 family is significantly reduced, and this is associated with shorter survival times in patients. However, the mechanisms leading to let-7 downregulation in cancer are still largely unclear. Since an alteration in copy-number is one of the causes of gene deregulation in cancer, we examined copy number alterations of the let-7 family in 2,969 cancer specimens from a high-resolution SNP array dataset. We found that there was a reduction in the copy number of let-7 genes in a cancer-type specific manner. Importantly, focal deletion of four let-7 family members was found in three cancer types: medulloblastoma (let-7a-2 and let-7e), breast cancer (let-7a-2), and ovarian cancer (let-7a-3/let-7b). For example, the genomic locus harboring let-7a-3/let-7b was deleted in 44% of the specimens from ovarian cancer patients. We also found a positive correlation between the copy number of let-7b and mature let-7b expression in ovarian cancer. Finally, we showed that restoration of let-7b expression dramatically reduced ovarian tumor growth in vitro and in vivo. Our results indicate that copy number deletion is an important mechanism leading to the downregulation of expression of specific let-7 family members in medulloblastoma, breast, and ovarian cancers. Restoration of let-7 expression in tumor cells could provide a novel therapeutic strategy for the treatment of cancer.

Figure 1. The genomic locus harboring the let-7a-3/let-7b cluster shows copy number deletions in breast and ovarian cancers.

The segmented raw data from the SNP arrays (breast cancer, n = 293; ovarian cancer, n = 110; myeloproliferative disorder, n = 215) was retrieved from the Tumorscape database, analyzed, then visualized by the Integrative Genomics Viewer. Left panel: whole genome wide view of the copy number profiles from myeloproliferative disorder, breast cancer, and ovarian cancer (genomic locations are on the left; tumor specimens are across the top). Red represents amplification and blue represents deletion. Right panel: Copy-number profiles of chromosome 22 in the region of the let-7a-3/let-7b cluster.

Figure 2. Members of the let-7 family show copy number deletions in medulloblastoma, breast, and ovarian cancers.

Summary of DNA copy number alterations of the let-7 family in 14 types of human cancers (n = 2,969). The let-7 family is made up of thirteen members located at eight loci of the human genome. Many of them, such as let-7a-1, let-7f-1 and let-7d, cluster together (A). Low q-values (upper threshold = 0.25) suggest that amplifications/deletions at this locus are significant and enriched by selective pressures. Dark green represents focal deletion of the let-7 family.

Figure 3. Copy number alteration of let-7b is positively correlated with mature let-7b expression in ovarian cancer.

Level 3 data (segmented SNP array data and normalized miRNA array data) was retrieved from TCGA. A total of 537 tumors with both SNP and miRNA arrays data were identified. A. Left panel: whole genome wide view of copy number profiles in ovarian cancer (genomic locations are on the left; tumor specimens are across the top). Red represents amplification and blue represents deletion. Right panel: Copy-number profiles from chromosome 22 in the region of the let-7a-3/let-7b cluster. B. Heat map of mature let-7 family expression levels in matched TCGA specimens. The samples are arranged in the same order as the SNP data. C. Correlations between let-7b DNA copy number and mature let-7b expression levels in ovarian cancer from the TCGA dataset. D. Correlations between let-7b DNA copy number and expression levels of mature miRNA of other let-7 family members in ovarian cancer from the TCGA dataset.

Figure 4. Restoration of let-7b expression significantly reduces ovarian tumor growth in vitro.

The let-7b mimic and control oligo (30 nM) were transfected into the A2780 (A), 2008 (B) and HOSE (C) cells by lipofectamine. The cell growth was monitored by a MTT assay.

Figure 5. Restoration of let-7b expression significantly reduces ovarian tumor growth in vivo. A.

Timeline of transplantation of tumor cells by intraperitoneal injection, treatment, imaging, and collection of tumor samples in the orthotopic late stage ovarian cancer mouse model. B. Tumor nodes collected from each mouse three days after the last treatment. C. Summary of the weights of the tumor nodes from each mouse. D. Endogenous let-7b activity in each mouse as monitored by a let-7b luciferase sensor. E. Summary of the luciferase intensity during the treatment.