S U M M A R Y Primary cilia (PC) are solitary, sensory organelles that are critical for several signaling pathways. PC were detected by immunofluorescence of cultured cells and breast tissues. After growth for 7 days in vitro, PC were detected in ?70% of breast fibroblasts and in 7-19% of epithelial cells derived from benign breast (184A1 and MCF10A). In 11 breast cancer cell lines, PC were present at a low frequency in four (from 0.3% to 4% of cells), but were absent in the remainder. The cancer cell lines with PC were all of the basal B subtype, which is analogous to the clinical triple-negative breast cancer subtype. Furthermore, the frequency of PC decreased with increasing degree of transformation/progression in the MCF10 and MDA-MB-435/LCC6 isogenic models of cancer progression. In histologically normal breast tissues, PC were frequent in fibroblasts and myoepithelial cells and less common in luminal epithelial cells. Of 26 breast cancers examined, rare PC were identified in cancer epithelial cells of only one cancer, which was of the triple-negative subtype. These data indicate a decrease or loss of PC in breast cancer and an association of PC with the basal B subtype. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58:857-870, 2010)

Triple negative breast cancers (TNBC) lacking estrogen, progesterone and HER2 receptors account for 10–20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly-targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial-mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide we performed an in silico screen for PAH2 domain-binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects of SID peptide to block SIN3-PAH2 interaction with MAD, induce expression of CDH1 and ESR1 and restore tamoxifen sensitivity in MDA-MB-231 human and MMTV-Myc mouse TNBC cells in vitro. Treatment with selamectin or ivermectin led to transcriptional modulation of genes associated with EMT and maintenance of a cancer stem cell phenotype in TNBC cells. This resulted in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo. Underlining the potential of avermectins in TNBC, pathway analysis revealed that selamectin also modulated the expression of therapeutically-targetable genes. Consistent with this, an unbiased drug screen in TNBC cells identified selamectin-induced sensitization to a number of drugs, including those targeting modulated genes.

Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poorer survival in triple negative breast cancer (TNBC) patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need in the treatment of cancer. We reported that the selective inhibition of the PAH2 domain of SIN3A protein function markedly suppressed metastatic dissemination to the lungs in TNBC xenograft bearing mice. Here, we show that TNBC cell lines treated with Sin3 interaction domain (SID) decoy peptides that bind to PAH2 display a strong in vitro inhibition of transwell invasion. This is accompanied by actin cytoskeleton reorganization with increased cortical actin deposition and downregulation of known Wnt target genes that are associated with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Wnt pathway inhibition by SID decoy peptide was confirmed by decreased Wnt reporter activity and altered cytoplasmic localization of nuclear β-catenin. TGIF1, a transcription factor that modulates Wnt signaling and known to interact with the PAH2 domain of SIN3A, can be dissociated from the SIN3A complex by SID decoys. TGIF1 knockdown inhibits WNT target genes and in vitro cell invasion suggesting that TGIF1 might be a key target of the SID decoys to block tumor invasion. Taken together, targeting SIN3 function using SID decoys is a novel strategy to reverse invasion and the EMT program in TNBC translating into the inhibition of metastasis dissemination and eradication of residual disease.