A team from Ohio State University has published new data identifying microRNA-486 as a potent tumor suppressor in lung cancer, finding that the miRNA affects cellular proliferation and survival by regulating insulin growth factor and that it is at least partially dependent on another tumor suppressor — p53 — for its anti-tumor effects.
While miR-486 has been shown in a number of studies from different groups to be downregulated in lung cancer, with expression patterns identifiable in the blood and sputum of patients, "the actual physiological role of the microRNA — whether it is a tumor suppressor or an oncogene — has not been elucidated," OSU investigator and study co-author Patrick Nana-Sinkam told Gene Silencing News.
Although the researchers didn't initially set out to focus on the miRNA, they began looking at it more closely after conducting a high-throughput miRNA profiling study on tumor tissue samples from 81 patients with stage I non-small cell lung cancer patients as part of an ongoing effort to examine the roles of the non-coding RNAs in cancer.
"As expected, there were several of the usual suspects that were overexpressed or downregulated," including miR-21 and miR-126, he explained. "What was striking was that miR-486 was the [most downregulated] microRNA in tumor compared to adjacent uninvolved lung."
Given miR-486's low expression in diseased tissue, the researchers speculated that it may function as a tumor suppressor. Indeed, overexpressing the miRNA into two independent NSCLC cell lines resulted in a reduction in cellular proliferation, lower colony formation, and an ostensible increase in cell-cycle arrest and apoptosis, according to their study, which appeared in the Proceedings of the National Academy of Sciences. The miRNA also lowered the migratory capacity in one of the cell lines.
Having established an anti-tumor effect for miR-486 in vitro, the investigators then looked to identify the miRNA's targets. In silico prediction tools led them to several components of the insulin growth factor, or IGF, tyrosine kinase signaling pathway, specifically IGF1, IGFR1, and p85-alpha. Notably, the 3' UTRs of these targets contained sequences complementary to the miR-486 seed sequence.
They then used siRNAs to selectively knock down IGFR1 and p85-alpha in NSCLC cell lines, and discovered that inhibition of IGFR1 caused a signiﬁcant increase in cells in G1 phase, while blocking IGF1R led to a "slight increase" in cells in sub-G0 phase, they wrote in PNAS. "We next showed that knockdown of p85-alpha and IGF1R, both alone and in combination, decreased migratory capacity of cancer cells."
Taken together, these data indicate that the effects of miR-486 on cell cycle progression and cell migration are partially mediated through miR-486 targeting of p85-alpha and IGF1R, the researchers added.
Having observed a "trend toward p53 wild-type cell lines having higher miR-186 expression than other non-wild-type cell lines" in preliminary studies, the researchers attempted to see if p53 expression was somehow involved in the miRNA's induction.
Two p53 wild-type lung cancer cell lines, along with a p53-null cell line, were treated with a small molecule inhibitor of the tumor suppressor, resulting in a "significant induction" of the miRNA's expression in both wild-type cell lines but not the mutant p53 line. The expression of p53 was then knocked down using siRNAs, which attenuated miR-486 induction in the wild-type cells.
The research team also found that overexpression of miR-486 induced apoptosis in the p53 wild-type cell lines, but not the p53-null line. Meanwhile, knockdown of p53 via RNAi decreased the induction in apoptosis mediated by miR-486 overexpression, suggesting that p53 activity is necessary for the miRNA's effects on apoptosis, according to the scientists.
"Moreover, forced expression of miR-486 resulted in increased expression of p53 upregulated modulator of apoptosis — a pro-apoptotic p53 target — and this induction was attenuated by p53 knockdown, supporting our ﬁnding that p53 is involved in miR-486–induced apoptosis," they noted in their paper.
To Nana-Sinkam, this association between miR-486 and p53 was surprising. "We really didn't expect to see one tumor suppressor — p53 — really driving another tumor suppressor — in this case, a microRNA," he noted.
To extend their findings into in vivo studies, the researchers next subcutaneously injected lung cancer cell lines with or without overexpressing miR-486 into the flanks of nude mice. Within 24 hours of injection, overexpression of the miRNA caused a "substantial reduction in tumor volume."
Further, one of the miR-486 cell lines did not form any detectable xenograft tumors in comparison to tumor-forming control cells, and Western blot analysis showed that expression of IFGR1 and p85-alpha were reduced in the miRNA-expression tumors.
While Nana-Sinkam said that the data described in PNAS point to the potential of miR-486 as a therapeutic agent itself, in the near term it could possibly be used as a clinical biomarker.
"We know that miR-486 appears to target several components of IGF signaling," he said. Given that clinical studies of cancer drugs directed against IGF signaling have had mixed results, "we start to wonder if it possible to use miR-486 as biomarkers that distinguish potential [therapy] responders from non-responders."
At the same time, the OSU team's findings further support the possibility that the miRNA could be used diagnostically.
Studies to date that indicate miR-486 expression patterns in biofluids can distinguish individuals with lung cancer from those who don't have the disease are encouraging, but have been limited in size, he noted. "We'd certainly like to expand upon that in larger cohorts to really see if [the findings] hold true," he said.