In the present study, we define a schwannoma-typical miRNA signature by miRNA microarray expression profiling of human vestibular schwannomas as compared to control nerves. This signature includes 12 miRNAs that are deregulated in most schwannoma tumor samples. Out of these 12 miRNAs, 8 were confirmed to be significantly upregulated in schwannomas (5–20-fold); and 4 miRNAs downregulated (5–12-fold). In this study, we focused on one of the most downregulated miRNA, miR-7, and found that overexpression of miR-7 inhibited schwannoma cell growth both in culture and in xenograft and orthotopic schwannoma tumor models in vivo. Our studies describe a novel target of miR-7, which directly targeted the sequences in the 3’UTR of the Ack1 mRNA with upregulation of miR-7 decreasing levels of Ack1 mRNA and protein in schwannoma cells. A significant inverse correlation was also found between miR-7 downregulation and Ack1 and Pak1 upregulation in human schwannoma tumor samples compared to control nerve tissue.
The overexpression or genomic amplification of Ack1 has been recently shown for breast, esophageal, lung, ovarian, pancreatic and prostate cancer (20
). In prostate cancer, Ack1 stimulates tumorigenesis in part by negatively regulating the proapoptotic tumor suppressor, the WW domain containing oxidoreductase (Wwox) (33
). Ack1 interacts with Wwox and triggers its ubiquitination and degradation. The same study also elucidated an oncogenic role of Ack1 in vivo,
with Ack1 overexpression promoting anchorage-independent growth and tumor formation in vivo
). It remains to be investigated how upregulation of Ack1 by decreased miR-7 contributes to schwannoma tumorigenesis.
Based on the relative fold increase as assessed by qRT-PCR assays, the most upregulated miRNAs in schwannomas were let-7d (about 22-fold), miR-451 (about 17-fold), and miR-23b (about 15-fold). The let-7 family has been the most studied of the potential “tumor suppressor” miRNAs and contains 11 family members (reviewed in 21
). This family acts as tumor suppressors to control several oncogenic pathways, including the Ras pathway (12
), as well as oncogenes, such as c-Myc (34
). The second most upregulated miRNA in schwannomas, miR-451 was also recently shown to function as a potential tumor suppressor in human gastric and colon cancer cells, with its overexpression decreasing proliferation and increasing response to ionizing radiation in culture (22
). In human malignant prostate cancers, miR-23a and miR-23b were shown to be downregulated compared to normal prostate tissues (35
). In summary, several previously known downregulated “tumor suppressor miRNAs” in malignant tumors, such as let-7d, miR-451, miR-23a, and miR-29 were found to be upregulated in schwannomas. Based on these observations and given the fact that let-7d, let-7b, and let-7g tumor suppressor miRNAs are also upregulated in benign meningiomas (13
), it seems likely that these miRNAs may function as a pivotal point in tumor progression between benign and malignant states by regulating certain oncogenic pathways.
In conclusion, growth inhibitory/apoptotic effects of elevated miR-7 levels were found in human primary and immortalized schwannoma cells, as well as in a non-immortalized mouse schwannoma cell line. miRNA expression profiling and functional studies of miR-7 in schwannoma tumor tissue and cells in culture suggest that miR-7 acts as a potential tumor suppressor in schwannomas, at least in part through direct targeting of IRS-2 (15
), Ack1, Pak1, and EGFR. Studies supporting a role for Pak1 (36
), EGFR (38
), and Ack1 (this study) activation/overexpression in schwannoma growth, suggests alternative strategies and rationale for the development of new therapies for these tumors based on overexpression of miR-7 or inhibition of Ack1, Pak1, and EGFR pathways. Given the fact that schwannomas, as many other cancers, are not always responsive to anti-EGFR treatment (39
), our study suggests that Pak1 and/or Ack1 may prove critical therapeutic targets for schwannomas.