Induction of endoplasmic reticulum stress is associated with the anti-tumor activity of monepantel across cancer types

Abstract

Background: Monepantel is an anti-helminthic drug that also has anti-cancer properties. Despite several studies over the years, the molecular target of monepantel in mammalian cells is still unknown, and its mechanism-of-action is not fully understood, though effects on cell cycle, mTOR signalling and autophagy have been implicated.

Methods: Viability assays were performed on >20 solid cancer cell cells, and apoptosis assays were performed on a subset of these, including 3D cultures. Genetic deletion of BAX/BAK and ATG were used to establish roles of apoptosis and autophagy in killing activity. RNA-sequencing was performed on four cell lines after monepantel treatment, and differentially regulated genes were confirmed by Western blotting.

Results: We showed that monepantel has anti-proliferative activity on a broad range of cancer cell lines. In some, this was associated with induction of apoptosis which was confirmed using a BAX/BAK-deficient cell line. However, proliferation is still inhibited in these cells following monepantel treatment, indicating cell-cycle disruption as the major anti-cancer effect. Previous studies have also indicated autophagic cell death occurs following monepantel treatment. We showed autophagy induction in multiple cell lines; however, deletion of a key autophagy regulator ATG7 had minimal impact on monepantel's anti-proliferative activity, suggesting autophagy is associated with, but not required for its anti-tumour effects. Transcriptomic analysis of four cell lines treated with monepantel revealed downregulation of many genes involved in the cell cycle, and upregulation of genes linked to ATF4-mediated ER stress responses, especially those involved in amino-acid metabolism and protein synthesis.

Conclusions: As these outcomes are all associated with mTOR signalling, cell cycle and autophagy, we now provide a likely triggering mechanism for the anti-cancer activity of monepantel.

Keywords: ER stress; autophagy; cell cycle; mTOR; monepantel.

FIGURE 1

Monepantel treatment reduces cell viability across a range of cancer types. Representative cell lines were treated with monepantel for 24–120 h then viability measured using CTG assays. Values shown are mean ± SEM of n = 3 experiments. Data for all cell lines tested are available in Figures S1 and S2.

FIGURE 2

Monepantel induces apoptosis in some but not all cell lines. (A) Representative cell lines were treated for 24–120 h with the indicated doses of monepantel and then analyzed for apoptosis induction by FACS following staining with Annexin V and PI. Values shown are mean ± SEM of n = 3 experiments. (B) HCT 116 cells show evidence of apoptosis, but this is significantly attenuated in the absence of BAX and BAK. Values shown are mean ± SEM of n = 4 experiments. (C) Spheroid cultures of LM‐MEL‐28, but not A549 cells undergo apoptosis following treatment with 25 μM monepantel for 168 h (***p = 0.006). (D) Representative images of the spheroids after treatment with vehicle or monepantel. (E) Western blot confirming deletion of BAX and BAK in BAX ^−/‐ BAK ^−/− HCT 116 colorectal cancer cells. β‐Actin was used as a loading control. (F) Deletion of BAX and BAK has only a minor impact on cell viability in CTG assays following monepantel treatment in HCT 116 cells. Values shown are mean ± SEM of n = 3 experiments. *p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, ****p < 0.0001.

FIGURE 3

Autophagy is not required for the anti‐proliferative activity of monepantel. (A–C) Western blots for autophagy markers of indicated cell lines following monepantel treatment at indicated timepoints and doses. Quantitation of LC3B blots is shown in Figure S5. (D, E) Western blots showing deletion of ATG7 following doxycycline (DOX) treatment in indicated cell lines. β‐actin was used as a loading control in all Western blots. (F) CTG viability assays on the indicated cell lines that were either wild‐type (ATG7 ^+/+ ) or KO (ATG7 ^−/− ). Values shown are mean ± SEM of n = 3 or 4 experiments. G, H) FACS‐based apoptosis assays on the indicated cell lines that were either ATG7 wild‐type or KO. Values shown are mean ± SEM of n = 3 experiments. *p = 0.01–0.05, **p = 0.001–0.01, ***p = 0.0001–0.001, ****p < 0.0001.

FIGURE 4

Transcriptomics analysis of four cell lines following monepantel treatment. (A) Venn diagram showing unique and overlapping differentially expressed genes (monepantel treated versus vehicle control) for all four cell lines. (B) Barplots for the top 25 GO terms enriched in the differentially expressed genes up‐regulated (blue bars) and down‐regulated (red bars) in all four cell lines. (C) Genes related to ER‐stress including those involved in amino acid synthesis and metabolism, apoptosis and mTOR signaling upregulated in all four cell lines. (D) Network map showing connections between major GO and KEGG terms for upregulated differentially expressed genes in all four cell lines.

FIGURE 5

Gene expression analysis of the three cell lines (A549, LM‐MEL‐28, OVCAR‐3) sensitive to monepantel treatment. (A) Heatmap showing differentially expressed genes in the sensitive cell lines but not HOSE 6‐3. Relative expression levels (Z‐scores) of genes are shown in the heat maps, color‐coded according to the legend. Rows are scaled to have a mean of 0 and an s.d. of 1. (B) Barplots for the top 10 GO terms enriched in the differentially expressed genes up‐regulated (blue bars) and downregulated (red bars) in the three sensitive cell lines but not in the HOSE 6‐3 cell line. (C) Barplots for the top KEGG terms enriched in the differentially expressed genes downregulated (red bars) in the three sensitive cell lines but not in cell line HOSE 6‐3.

FIGURE 6

Validation of transcriptomics data. (A) Cell cycle analysis shows an increase in the G0/G1 population following monepantel treatment in OVCAR‐3 cells. Values shown are mean ± SEM of n = 2 experiments. (B) Western blot analysis shows an increase in NOXA levels in the four indicated cell lines, consistent with the transcriptomics data. (C) Western blot analysis shows an increase in 4E‐BP1 (hypophosphorylated form) in the four indicated cell lines, consistent with the transcriptomics data. (D) Western blots showing BiP and CHOP proteins are upregulated following monepantel treatment, consistent with cells undergoing ER stress. (E) MEFs deficient for Atf4 (Atf4 ^−/− ) are more sensitive to monepantel treatment compared to their wild‐type (Atf4 ^+/+ ) counterparts. Cell lines were treated with monepantel for 24–120 h then viability measured using CTG assays. Values shown are mean ± SEM of n = 3 experiments (**p = 0.001–0.01).

FIGURE 7

Schematic showing how monepantel activates multiple pathways to elicit its anti‐cancer effects. Processes in red circles are downregulated while those in green circles are upregulated.