In vitro activity: ML327 de-represses E-cadherin transcription, partially reverses EMT, and inhibits cancer cell invasiveness and tumor cell migration in vitro and in vivo. Induction of E-cadherin mRNA expression by ML327 treatment does not require de novo protein synthesis. RNA sequencing analysis revealed that ML327 treatment significantly alters expression of over 2,500 genes within three hours in the presence of the translational inhibitor, cycloheximide. Network analysis reveals Hepatocyte Nuclear Factor 4-alpha (HNF4α) as the most significant upstream transcriptional regulator of multiple genes whose expressions were altered by ML327 treatment. Further, small interfering RNA-mediated depletion of HNF4α markedly attenuates the E-cadherin expression response to ML327. In summary, ML327 represents a valuable tool to understand mechanisms of EMT and may provide the basis for a novel targeted therapeutic strategy for carcinomas.

Kinase Assay: Treatment with ML327 induces an elongated morphology in neuroblastoma cells. BE(2)-C cells treated with ML327 demonstrates G1 cell cycle arrest with a concordant decrease in S phase population, and a significant increase in the sub G0 population. ML327 induces the expression of CDH1 in all seven of the neuroblastoma cell lines with a 50 to 1,400-fold induction of CDH1 mRNA expression. ML327 blocks the expression of MYC family of oncogenic transcription factors in all tested neuroblastoma cell lines. Immunoblotting time course demonstrates early repression of N-MYC expression within 2 h of treatment with ML327 (10 μM). p53 levels are also suppressed by treatment with ML327. ML327-pretreated cells demonstrates reduced proliferative potential in both tetrazolium-based (p<0.0001) and adherent 2D colony formation (41 vs. 400; p<0.0001). ML327 reduces SW620inv cell invasion through Matrigel by ~60% and reduces H520 cell invasion by ~30% in these in vitro assays. ML327 partially restores E-cadherin expression at the plasma membrane in NMuMG cells induced to undergo Epithelial-to-Mesenchymal Transition (EMT) by TGF-β1 treatment.

Cell Assay: Cells are seeded onto 96-well plates at equivalent density (3,000 to 10,000 depending upon cell line), permitted to attach overnight, and treated with either ML327 (10 μM) or vehicle. Daily absorbance measurements (450 nm) using the cell counting kit are obtained. For estimation of IC50 values, cells are plated at equal density, permitted to attach, and baseline absorbance is obtained using cell counting kit. Cells are then treated with varying doses of ML327 (0.1 to 30 μM) and cell viability is measured 72 h after treatment.