Scientists Identify "Feed-Forward Loop" Involving Micro-RNA and Sp1 in Multiple Myeloma

For the first time, scientists have identified a "feed-forward loop" consisting of a gene, micro-RNA, and transcription factor implicated in both multiple myeloma and Waldenstrom's macroglobulinemia, according to an article published this month in the Blood Cancer Journal.

Nikhil Munshi, MD

For the first time, scientists have identified a "feed-forward loop" consisting of a gene, micro-RNA, and transcription factor implicated in both multiple myeloma and Waldenström's macroglobulinemia, according to an article published this month in the Blood Cancer Journal.1

The loop, which links the gene Sp1 with the micro-RNA miR-23b and the transcription factor c-Myc, was found to lead to down-regulation of miR-23b in multiple myeloma.

The paper summarized results of experiments performed utilizing a large database of hundreds of multiple myeloma cell lines and fewer than 100 Waldenstrom's cell lines. This sample population also included peripheral blood mononuclear cells obtained from healthy volunteers and online databases. Similar results were obtained in experiments in mice, the paper said.

"Whenever we over-express these micro-RNA, we achieve inhibition of myeloma cell growth," explained Mariateresa Fulciniti, PhD, senior scientist, Dana Farber Cancer Institute at Harvard Medical School, lead author on the paper in an interview withTargeted Oncology.

"All three components of the loop can regulate each other, but in cancer, these loops are dysfunctional, which could be because of any one of the components. But if you bring about control, then you can reset the cell and it will behave more normally," she added.

"Close to 90% of myeloma patients show down-regulated expression of Sp1," added Nikhil Munshi, MD, professor of medicine, Dana Farber Cancer Institute, in an interview withTargeted Oncology. The gene has also been implicated in breast, colon cancer, and lymphoma.

Transcription factor genes have previously been implicated in drug resistance, according to the paper.

A micro-RNA is a segment of small, non-coding RNAs, 20 to 22 nucleotides in length. About 2000 miRNAs have been identified, Munshi said. According to the paper, like other miRNAs, 23b has been shown to up-regulated in some cancers, but down-regulated in others. For example, micro-RNA 23b (miR-23b) is inactive in prostate cancer and glioblastoma.

The researchers previously discussed the role of the gene Sp1 in myeloma2and Waldenström's3, but this is the first time the interconnection with the other elements of the loop has been described.

"Now the thing is to see which genes these micro-RNA control," Fulciniti said.

Pre-clinical Research

Fulciniti and colleagues "first identified miR-23b as one of the most significant negative regulator of Sp1 expression (data not shown)," the article said. "Consistently, miR-23b mimics transfection in BCMW1 and NCI-H929 cells reduced Sp1 protein expression as well as the levels of known Sp1 transcriptional targets."

Researchers carried out a variety of pre-clinical experiments involving molecular imaging by means of the firefly luciferase reporter gene, as well as artificial alteration of cancer cell lines, by means of cloning, to overexpress miRNA-23b.

The paper described an experiment in which researchers examined data from cells taken from 38 multiple myeloma patients and compared them with normal cells. Expression of miRNA was decreased in the myeloma cells compared to normal cells (P=.00016). Other experiments showed that cancer cell growth at seven days in myeloma cell lines from the National Cancer Institute (NCI) was reduced when the cells had been engineered to stably express miR-23b compared to myeloma cells in which the micro-RNA was not stably expressed (P=.0032).

Researchers also looked at how miR-23b over-expression affected the formation of cancer cell colonies. At day 21, they found that average colony numbers in a line of myeloma cells from the NCI which had been engineered to over-express miR-23b was reduced compared to normal multiple myeloma cells (P=.0005). A similar experiment carried out with Waldenstrom's cells found similar results (P=.0018).

"Upon silencing of myc with specific siRNAs, we observed increased miR-23b expression in a dose-dependent way. On the other hand, upregulation of c-myc in the c-myc-negative U266 cell line decreased miR-23b expression," the article said.

Additionally, an experiment in mice injected with myeloma cells that had been altered to over-express miR-23b found that tumor growth was decreased in comparison to control mice after 15 days (P=.03).

The results indicate that targeted miR-23b-replacement therapy might be an effective treatment for the cancers.

"Additional genomic correlates need to be considered for an integrated oncogenomic analysis," the paper said.

References

  1. Fulciniti M, Amodio N, Bandi R, et al. miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.Blood Cancer Journal(2016) 6, e380; doi:10.1038/bcj.2015.106 www.nature.com/bcj http://www.nature.com/bcj/journal/v6/n1/full/bcj2015106a.html
  2. Fulciniti M, Amin S,, Nanjappa P, et al. Significant Biological Role of Sp1 Transactivation in Multiple Myeloma.Clin Cancer Res; 17(20) October 15, 2011. Published OnlineFirst August 19, 2011; DOI: 10.1158/1078-0432.CCR-11-1036
  3. Fulciniti M, Amodio N, Bandi R, et al. MYD88-independent growth and survival effects of Sp1 transactivation in Waldenström macroglobulinemia.BLOOD, 24 April 2014 x Volume 123, Number 17 2673. 2014 123: 2673-2681 doi:10.1182/blood-2014-01-550509 originally published online March 12, 2014