Autophagy inhibition synergistically enhances anticancer efficacy of RAMBA, VN/12-1 in SKBR-3 cells, and tumor xenografts

Abstract

VN/12-1 is a novel retinoic acid metabolism blocking agent discovered in our laboratory. The purpose of the study was to elucidate the molecular mechanism of anticancer activity of VN/12-1 in breast cancer cell lines and in tumor xenografts. We investigated the effects of VN/12-1 on induction of autophagy and apoptosis in SKBR-3 cells. Furthermore, we also examined the impact of pharmacologic and genomic inhibition of autophagy on anticancer activity of VN/12-1. Finally, the antitumor activity of VN/12-1 was evaluated as a single agent and in combination with autophagy inhibitor chloroquine in an SKBR-3 mouse xenograft model. Short exposure of low dose (<10 μmol/L) of VN/12-1 induced endoplasmic reticulum stress, autophagy, and inhibited G(1)-S phase transition and caused a protective response. However, a higher dose of VN/12-1 initiated apoptosis in vitro. Inhibition of autophagy using either pharmacologic inhibitors or RNA interference of Beclin-1 enhanced anticancer activity induced by VN/12-1 in SKBR-3 cells by triggering apoptosis. Importantly, VN/12-1 (5 mg/kg twice weekly) and the combination of VN/12-1 (5 mg/kg twice weekly) + chloroquine (50 mg/kg twice weekly) significantly suppressed established SKBR-3 tumor growth by 81.4% (P < 0.001 vs. control) and 96.2% (P < 0.001 vs. control), respectively. Our novel findings suggest that VN/12-1 may be useful as a single agent or in combination with autophagy inhibitors for treating human breast cancers. Our data provides a strong rationale for clinical evaluation of VN/12-1 as single agent or in combination with autophagy inhibitors.

Figure 1. Effect of VN/12-1 on the growth of human breast cancer cells and ERS markers in vitro

(A) Chemical structures of VN/12-1 and CHL (B) VN/12-1 inhibits the growth of MCF-7 (green), SKBR-3 (blue), MDA-MB-231 cells (red), MCF-10A (black). Curves generated from an MTT assay after 96 h exposure to VN/12-1. Points, mean of replicates from three independent experiments; bars, SE. Solid line, best-fit sigmoidal-dose response (variable slope). (C) Cells were exposed to 10 µM VN/12-1 for time points shown. After each time point, the compound was washed away and cells were maintained in normal growth medium for 96 hrs, and cell viability was measured with an MTT assay.* p < 0.05, ** p < 0.01. (D(a)–D(b)) Effect of VN/12-1 on ERS markers. Cells were treated with indicated concentrations of VN/12-1 for 6 h (D(a)) or 24 h (D(b)), whole cell lysates were tested for BiP and p-eIF2α. T-Thapsigargin (20 µM) was used as a positive control. Total eIF2α was used as loading control.

Figure 2. VN/12-1 induces the formation of autophagosomes

(A–B) Transmission electron microscopy images of VN/12-1-treated SKBR-3 cells. Cells were treated with ethanol (A) or 10 µM VN/12-1 (B (a), (b), (c)) for 24 h. (A and B(a)) represent comparison of nuclear and nucleolar morphology (arrow heads); (A and B(b)) represent normal versus VN/12-1-treated mitochondria (solid arrows in (A) vs arrow head in B(b)); B(a) VN/12-1-treated cell with vacuolated cytoplasm (solid arrow); B(b) VN/12-1-treated cell with dilated ER showing ER stress (solid arrow); and B(c) early (solid arrow) and late (arrow head) autophagosomes containing cellular organelles. (C) SKBR-3 were treated with ethanol (a) or 10 µM VN/12-1 (b) for 24 h. VN/12-1 induces LC3 aggregation (punctate staining– peculiar of autophagy formation as shown in figure 2C (b)).

Figure 3. VN/12-1 induces autophagy markers and cell cycle arrest in SKBR-3 cells

(A(a) and A(b)) Effect of VN/12-1 on autophagy markers. Cells were treated with indicated concentrations of VN/12-1 and CHL for 24 h, whole cell lysates were tested for LC3B, Beclin-1, p-p70S6K and p-Akt. β-actin and total Akt, total p70S6K were used as control. (B) Cells were treated with indicated concentrations of VN/12-1 and/or CHL for 24 h. Whole cell lysates were tested for ERS markers and cyclin D1. V-VN/12-1, C-CHL. The number following V or C indicates the concentration in µM. (C) SKBR-3 cells were treated with indicated concentrations of VN/12-1 and IC20 concentrations of (C(a)) CHL, (C(b)) 3-Methyladenine (3-MA) for 96 h in an MTT assay. Columns are the mean of viable cells in three experiments; bars, SE. ** p < 0. (D) Cell were transfected with si-Beclin-1 or si-Scrambled as described above. After 72 hours, they were treated with indicated concentrations of VN/12-1 and cell viability was assessed by MTT assay as described above. * P < 0.05, ** p < 0.01.

Figure 4. VN/12-1 (in combination with CHL) induces caspase dependent apoptosis

(A) SKBR-3 cells were treated with indicated concentrations of VN/12-1 or CHL for 24 h and protein expression of cleaved PARP and Bad was determined. V-VN/12-1, C-CHL. The number following V or C indicates the concentration in µM (B). The percent of late apoptotic/necrotic cells as determined by Annexin V-FITC and PI positive staining by flow cytometry. **p < 0.01. SKBR-3 cells were treated with indicated concentrations of VN/12-1 or CHL for 24 h. % cells stained with FITC/PI was calculated using flow cytometry. (C) Indicated concentrations of VN/12-1, CHL and/or 30 µM Z-vad-fmk used in an MTT assay (as described in Methods section) experiment to determine the cell viability in SKBR-3 cells. Columns are the mean of three experiments; bars, SE. * p < 0.05, ** p < 0.01.

Figure 5. VN/12-1 (alone or in combination with CHL) inhibits the growth of SKBR-3 xenografts

(A) The effect of VN/12-1 (2 doses – 2.5 and 5 mg/kg twice a week), ATRA (5 mg/kg twice a week) alone or in combination with CHL (50 mg/kg twice a week) were evaluated in a SKBR-3 xenograft model in female SCID mice. Mice (n = 8) were injected subcutaneously. Tumors were measured twice a week. (A(a)) Arithmetic tumor means and (A(b)) geometric means was plotted against time. (A(b)) has logarithmic scale on y-axis. P-values are indicated separately in supplementary Table S4 (B) Representative tumors from the 8 groups (C(a)) Mean tumor weights taken upon euthanizing all mice and collecting tumors. *p < 0.05, **p < 0.01. Data are mean (± s.e.). (C(b)) mean body weights. Mice were weighed once a week for the duration of the study. (D) Western blot analysis of protein expression in SKBR-3 tumors taken from mice. Autophagy marker (LC3B), ERS marker (CHOP), Cell cycle marker (cyclin D1), Apoptosis markers (Bad, PARP cleavage) were probed.

Figure 6. Mechanisms of action of VN/12-1 in combination with CHL

VN/12-1 induces ER stress. This results in inhibition of protein translation, downregulation key cell cycle proteins such as cyclin D1 and arrest in G1-S phase cell cycle transition. Addition of CHL enhances the ER stress and switches it from reversible (protective) pathway to irreversible (apoptotic) pathway by CHOP upregulation. VN/12-1 inhibits PI3K-Akt-mTOR pathway and activates autophagy. Inhibition of VN/12-1-mediated autophagy by CHL potentiates apoptosis by activation of caspase 9 and PARP cleavage.