4-Hydroxytamoxifen induces autophagic death through K-Ras degradation

Abstract

Tamoxifen is widely used to treat estrogen receptor-positive breast cancer. Recent findings that tamoxifen and its derivative 4-hydroxytamoxifen (OHT) can exert estrogen receptor-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in estrogen receptor-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNST) where estrogen is not involved. In this study, we gained insights into the estrogen receptor-independent cytotoxic effects of OHT by studying how it kills MPNST cells. Although caspases were activated following OHT treatment, caspase inhibition provided no protection from OHT-induced death. Rather, OHT-induced death in MPNST cells was associated with autophagic induction and attenuated by genetic inhibition of autophagic vacuole formation. Mechanistic investigations revealed that OHT stimulated autophagic degradation of K-Ras, which is critical for survival of MPNST cells. Similarly, we found that OHT induced K-Ras degradation in breast, colon, glioma, and pancreatic cancer cells. Our findings describe a novel mechanism of autophagic death triggered by OHT in tumor cells that may be more broadly useful clinically in cancer treatment.

Fig.1

OHT triggers autophagic death in MPNST cells. (A) OHT-treated cells demonstrated a concentration-dependent increase in levels of caspase-3 like activity (48 hours). * indicates p<0.05 relative to untreated cells. However, broad caspase inhibition with BAF (50μM, 1hour pre-treatment) (B) did not provide protection from OHT-induced death (48 hours). Exposure to OHT (48 hours) triggered (C) a concentration-dependent increase in steady state levels of LC3-II, and (D) an increase in autophagic flux. Cells transfected with siRNA against Atg7 demonstrated (E) unchanged levels of LC3-II following OHT treatment (48 hours) and (F) partial protection from OHT-induced cytotoxicity (72 hours). * indicates p<0.05 relative to cells transfected with N.T siRNA.

Fig.2

OHT triggers K-Ras degradation. Following OHT treatment (48 hours), (A) a concentration-dependent decrease was observed in K-Ras levels while (B) H- and (C) N-Ras levels remained unchanged. (D) Whole cell lysates from OHT-treated cells demonstrated a time-dependent decrease in K-Ras levels in the presence of the protein synthesis inhibitor CHX (100μM, 1hr pre-treatment). (E) OHT exposure caused a slight decrease (24hours) followed by an increase (48 hours) in levels of phosphorylated S6 protein. Rapamycin treatment was used as a positive control. (F) Co-immunoprecipitation experiments indicated no change in the interaction between eIF4E and 4EBP1 following OHT treatment. Amino acid starvation was used as a positive control.

Fig.3

OHT treatment inhibits MAPK signaling. Whole cell lysates from OHT-treated cells demonstrate a time-dependent decrease in (A) levels of phospho-p44/42 and (B) phospho-JNK. (C) Treatment with the PKC inhibitor R031-8220 (10μM, 24 hours) and OHT (10μM, 24 hours) caused a decrease while the calmodulin inhibitor W13 (15mg/ml, 24 hours) had no effect on steady state K-Ras levels. (D) Whole cell lysates from R031-8220-treated cells demonstrated a time-dependent decrease in K-Ras levels in the presence of the protein synthesis inhibitor CHX (100μM, 1hr pre-treatment).

Fig.4

Autophagy mediates OHT-induced death through K-Ras degradation. Inhibition of autophagy initiation using (A) 3-MA (5mM, last 12 hours of treatment) and (B) siRNA mediated Atg7 knockdown blocks the decrease in levels of K-Ras following OHT-treatment (48 hours). Cells expressing shRNA under tetracycline-inducible promoter were treated with dox. Following dox induction, (C) cells expressing control shRNA demonstrated no change in levels of K-Ras and cell death while (D) cells expressing K-Ras shRNA demonstrated a drop in K-Ras levels accompanied by an increase in cytotoxicity following OHT- treatment. * indicates p<0.05 relative to cells not subjected to dox exposure.

Fig.5

OHT triggers EGFR degradation. (A) Whole cell lysates from OHT-treated cells demonstrated a time-dependent decrease in EGFR levels in the presence of the protein synthesis inhibitor CHX (100μM, 1hr pre-treatment). (B) MPNST, (C, D and E) glioma and (F) breast cancer cell lines demonstrated a concentration-dependent decrease in EGFR levels following OHT treatment (48 hours).

Fig.6

OHT triggers K-Ras degradation in multiple tumor types. Whole cell lysates from (A) glioma, (B, C) MPNST, (D, E) pancreatic, (F, G) colon and (H, I) breast cancer cell lines demonstrated a decrease in K-Ras levels following OHT treatment (48 hours)

Fig.7

Mutant K-Ras resists OHT-induced degradation. Lysates from (A) breast and (B, C, D and E) colon cancer cell lines harboring a G13D mutation failed to demonstrate a decrease in K-Ras levels following OHT treatment (48 hours). (F) The colon cancer cell line T84 (G13D) was an exception.