Monoamine oxidase-A activity is required for clonal tumorsphere formation by human breast tumor cells

Abstract

BackgroundBreast tumor growth and recurrence are driven by an infrequent population of breast tumor-initiating cells (BTIC). We and others have reported that the frequency of BTIC is orders of magnitude higher when breast tumor cells are propagated in vitro as clonal spheres, termed tumorspheres, by comparison to adherent cells. We exploited the latter to screen > 35,000 small molecules to identify agents capable of targeting BTIC. We unexpectedly discovered that selective antagonists of serotonin signaling were among the hit compounds. To better understand the relationship between serotonin and BTIC we expanded our analysis to include monoamine oxidase-A (MAO-A), an enzyme that metabolizes serotonin.MethodsWe used the Nanostring technology and Western blotting to determine whether MAO-A is expressed in human breast tumor cell lines cultured as tumorspheres by comparison to those grown as adherent cells. We then determined whether MAO-A activity is required for tumorsphere formation, a surrogate in vitro assay for BTIC, by assessing whether selective MAO-A inhibitors affect the frequency of tumorsphere-forming cells. To learn whether MAO-A expression in breast tumor cells is associated with other reported properties of BTIC such as anticancer drug resistance or breast tumor recurrence, we performed differential gene expression analyses using publicly available transcriptomic datasets.ResultsTumorspheres derived from human breast tumor cell lines representative of every breast cancer clinical subtype displayed increased expression of MAO-A transcripts and protein by comparison to adherent cells. Surprisingly, inhibition of MAO-A activity with selective inhibitors reduced the frequency of tumorsphere-forming cells. We also found that increased MAO-A expression is a common feature of human breast tumor cell lines that have acquired anticancer drug resistance and is associated with poor recurrence-free survival (RFS) in patients that experienced high-grade, ER-negative (ER−) breast tumors.ConclusionsOur data suggests that MAO-A activity is required for tumorsphere formation and that its expression in breast tumor cells is associated with BTIC-related properties. The discovery that a selective MAO-A inhibitor targets tumorsphere-forming cells with potencies in the nanomolar range provides the first evidence of this agent’s anticancer property. These data warrant further investigation of the link between MAO-A and BTIC.

The Role of Serotonin in Breast Cancer Stem Cells

“…Consequently, MAO-A and MAO-B selective antagonists were evaluated using cell viability and sphere-forming assays. By contrast to our expectations, three selective MAO-A antagonists tested reduced cell viability and the frequency of sphere-forming cells derived from the two breast cancer cell lines (MCF-7 and HCC1954) that were tested [ 57 ]. MAO-B selective antagonists had no effect in the latter assays.…”

“…In advance of performing mechanistic studies with the antagonists, we determined whether MAO-A transcripts and protein were expressed in a panel of 10 human breast tumor cell lines comprising all clinical subtypes. MAO-A was not expressed in the majority of breast tumor cell lines when they were propagated in serum-containing media: however, its abundance at both the RNA and protein levels was dramatically increased when the tumor cells were cultured in chemically defined media as tumorspheres [ 57 ]. Hence, in vitro culture conditions conducive for maintaining a high BTIC frequency stimulates MAO-A expression.…”

“…In silico mining of gene expression profiles of breast tumor cell lines that overexpressed individual components of RTK signaling pathways revealed that epidermal growth factor, a component of the chemically defined media required for sphere formation, increased the abundance of MAOA transcripts [ 57 ]. Interestingly, MAOA transcript expression was also positively associated with resistance of breast tumor cell lines to both cytotoxic and targeted anticancer therapies.…”

“…Interestingly, MAOA transcript expression was also positively associated with resistance of breast tumor cell lines to both cytotoxic and targeted anticancer therapies. Moreover, like the expression of BTIC markers such as aldehyde dehydrogenase [ 71 ] and CD44 [ 72 ], increased MAOA transcripts expression is associated with reduced relapse-free survival in high-grade breast tumors of the TNBC clinical subtype [ 57 ]. These findings further strengthened the link between BTIC and MAO-A activity.…”

Antagonists of the serotonin receptor 5A target human breast tumor initiating cells

“…We subsequently demonstrated that structurally unrelated selective antagonists of other serotonin pathway components target mouse [17] and human BTIC [18] using multiple orthogonal assays. In short, antagonists of tryptophan hydroxylase 1 (TPH1), monoamine oxidase A (MAO-A), SERT and each of 5 of the 17 5-HTRs inhibited tumorsphere formation by each of six human breast tumor cell lines independent of the breast cancer subtype that they model [18,19]. A highly selective 5-HT5A antagonist (SB-699551) was the most potent of all the compounds tested in these assays.…”