NZ51, a ring-expanded nucleoside analog, inhibits motility and viability of breast cancer cells by targeting the RNA helicase DDX3

Abstract

DDX3X (DDX3), a human RNA helicase, is over expressed in multiple breast cancer cell lines and its expression levels are directly correlated to cellular aggressiveness. NZ51, a ring-expanded nucleoside analogue (REN) has been reported to inhibit the ATP dependent helicase activity of DDX3. Molecular modeling of NZ51 binding to DDX3 indicated that the 5:7-fused imidazodiazepine ring of NZ51 was incorporated into the ATP binding pocket of DDX3. In this study, we investigated the anticancer properties of NZ51 in MCF-7 and MDA-MB-231 breast cancer cell lines. NZ51 treatment decreased cellular motility and cell viability of MCF-7 and MDA-MB-231 cells with IC50 values in the low micromolar range. Biological knockdown of DDX3 in MCF-7 and MDA-MB-231 cells resulted in decreased proliferation rates and reduced clonogenicity. In addition, NZ51 was effective in killing breast cancer cells under hypoxic conditions with the same potency as observed during normoxia. Mechanistic studies indicated that NZ51 did not cause DDX3 degradation, but greatly diminished its functionality. Moreover, in vivo experiments demonstrated that DDX3 knockdown by shRNA resulted in reduced tumor volume and metastasis without altering tumor vascular volume or permeability-surface area. In initial in vivo experiments, NZ51 treatment did not significantly reduce tumor volume. Further studies are needed to optimize drug formulation, dose and delivery. Continuing work will determine the in vitro-in vivo correlation of NZ51 activity and its utility in a clinical setting.

Keywords: RNA helicase DDX3; ring-expanded nucleoside.

Figure 1. Characterization of MDA-MB-231 DDX3 knockdown cells

A. qRT-PCR analysis for the expression levels of DDX3 in MDA-MB-231-shDDX3 cell line and the parental MDA-MB-231 cells. Normalization was done by scoring for the expression levels of 36B4, a ribosomal gene. B. Immunoblot analysis demonstrating the decreased expression levels of DDX3 protein in MDA-MB-231-shDDX3 (231-shDDX3) cells relative to parental and vector control cells. C. Growth curve for MDA-MB-231-shDDX3, parental and vector control cells. D. Colony forming assay for MDA-MB-231-shLuc and MDA-MB-231-shDDX3 cells. E. Colony forming assay for MCF-7-shLuc and MCF-7-shDDX3 cells.

Figure 2. The effect of DDX3 knockdown of MDA-MB-231 on tumor growth, metastatic potential and tumor microenvironment

A. Graphical depiction of primary tumor volumes of the respective xenografts over an eight-week period. B. Post mortem H&E staining analysis of lungs from orthotopic primary tumor xenografts generated with either MDA-MB-231-shLuc or MDA-MB-231-shDDX3 cells. Red arrow points to the foci of the tumor cells in the lungs (p = 0.0377). C. Images of tumor vascular volume (VV) and permeability-surface area products (PS) obtained by MRI for control and MDA-MB-231shDDX3 cells. Red and green channels represent the distribution of VV and PS, respectively.

Figure 3. Structure of NZ51 and molecular modeling structure of NZ51 binding to DDX3

A. Chemical structure of NZ51. B. The energy-minimized complex of NZ51 and DDX3, indicating that NZ51 fits into ATP binding pocket. C. The aerial view of imidazole-diazepine ring system stacked with phenol ring of Tyr 200. Generation of a model of the interaction of drug with DDX3 is based on the reported crystal structure of DDX3 (PDB ID: 2I4I).

Figure 4. Effect of NZ51 on cell viability of normal breast cell lines and breast cancer cell lines as well as on cell cycle progression of MCF-7 and MDA-MB-231

A. Cell viability assay of normal immortalized breast cell lines and breast cancer cell lines incubated with different concentrations of NZ51 for 72 h at 0.001, 0.01, 0.1, 1, 5, 10 and 20 μM. B. Flow cytometry analysis determinations of the percentage of MCF-7 cancer cells in G1, G2/M, and S phase of the cell cycle during three consecutive days of treatment with NZ51 at 0, 4, 8, 12 μM. The latter concentrations are indicated under the bars of each graph. C. Flow cytometry analysis performed with MDA-MB-231 cancer cells.

Figure 5. In vitro wound-healing/scratch assay

A. MCF-7 cancer cells. B. MCF-7 cancer cells treated with 10 μM of NZ51. C. MDA-MB-231 cancer cells. D. MDA-MB-231 cancer cells treated with 10 μM of NZ51. Photomicrographs were obtained at the indicated time points using a 10X objective on a Nikon eclipse TS100 inverted microscope and recorded using NIS-Elements F 3.2 software.

Figure 6. NZ51 stabilizes DDX3 with inactivation of its function and is not affected by hypoxia

A. Immunoblot of MCF-7 and MDA-MB-231 total protein extracts from control and NZ51 treated scored for DDX3 and E-cadherin expression levels at the indicated post treatment times with β-actin as loading control. B. Photomicrographs of MDA-MB-231 cells before and after 72 h incubation with NZ51 under normoxic and hypoxic conditions. C. MCF-7 cells were either co-transfected with an E-cadherin promoter reporter construct (E2) along with a CMV-DDX3 expression vector or with only E2, followed by incubation with NZ51. Luciferase activity was estimated 24 h following NZ51 addition. The fold repression was calculated against the luciferase activity of E2 construct alone in MCF-7 cells. D. MTS assay results following NZ51 incubation under normoxic and hypoxic conditions for 72 hr.