Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Su-Jae Lee,Min-Jung Kim,Soo-Jung Woo,Sang-Gu Hwang,Sungkwan An,Gyesoon Yoon,Yung-Hyun Choi,Chang-Hwan Yoon,Jae-Seong Lee

doi:10.1074/jbc.m110.138958

Abstract

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Highlights

Ras GTPases act as molecular switches to transduce extracellular signals to the nucleus, where they regulate an overlapping set of cellular responses
We provide novel evidence that autophagy is an essential element in the induction of malignant transformation by oncogenic K-Ras and show that reactive oxygen species (ROS)2-mediated c-Jun N-terminal kinase (JNK) activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7
Inhibition of p38 MAPK clearly suppressed K-RasV12-induced autophagic vacuole formation (Fig. 6C) and anchorage-independent cell growth (Fig. 6C) as well as JNK activation. These results indicate that p38 MAPK signaling is involved in K-RasV12-induced ROS production and that JNK lies downstream of p38 MAPK-dependent ROS generation and plays a role in ATG5 and ATG7 up-regulation, autophagy, and malignant cell transformation

Summary

Introduction

Ras GTPases act as molecular switches to transduce extracellular signals to the nucleus, where they regulate an overlapping set of cellular responses. The phosphorylated forms of ERK, p38 MAPK, and JNK were up-regulated in cells overexpressing K-RasV12, indicating that these kinases are activated in response to oncogenic K-Ras. To further determine whether activation of MAPK might be required for K-RasV12-induced ROS generation and autophagy, we inhibited MAPK with specific pharmacological inhibitors or siRNAs. pretreatment with a JNK inhibitor or siRNA-mediated JNK knockdown suppressed K-RasV12-induced ATG5 and ATG7 expression.

Results

Conclusion