Loss of Smad4 promotes aggressive lung cancer metastasis by de-repression of PAK3 via miRNA regulation

Xiaohong Tan,Longying Tang,Lei Li,Jishuai Zhang,Xiao Yang,Shaofang Xie ,Nabeel Bardeesy,Feiran Gao,Qingwu Liu,Yanxiao Wang,Shihui Shen,Lei Ji,Junjiang Fu,Yun Liu,Jian Liu,Robb E Moses,Dianwen Song,Jinjin Xu,Kwok‐Kin Wong ,Lin Li,Lu Tong,Yanhui Xiao,Xiaotao Li

doi:10.1038/s41467-021-24898-9

Abstract

SMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generate a highly aggressive lung cancer mouse model bearing conditional KrasG12D, p53fl/fl LOF and Smad4fl/fl LOF mutations (SPK), showing a much higher incidence of tumor metastases than the KrasG12D, p53fl/fl (PK) mice. Molecularly, PAK3 is identified as a downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice is achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3′UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components is observed in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.

Highlights

SMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad[4] loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated
To investigate the contribution of Smad[4] LOF in lung cancer progression and metastasis in the context of conditional mouse lung cancer models, we utilized existing Cre/LoxP–controlled, genetically engineered mouse models with Kras (KrasG12D)[14], p53 LOF (p53fl/fl)[15], and Smad[4] LOF (Smad4fl/fl)[24,25] mutations to generate a cohort of lung tumors by nasal delivery of an adenovirus expressing Cre recombinase
Despite that Smad[4] interacts with R-Smads to potentiate TGFβ signaling, our results demonstrated that regulation of PAK3 by Smad[4] was not affected by the treatment of TGFβ or silencing Smad[3] (Supplementary Fig. 3a), suggesting a TGFβ-independent mechanism

Summary

Introduction

SMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad[4] loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Upregulation of PAK3 by Smad[4] LOF in SPK mice is achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543 These microRNAs (miRNAs) directly bind to the PAK3 3′UTR for blockade of PAK3 production, regulating lung cancer metastasis. We used adeno-Cre to conditionally activate a KrasG12D allele with concomitant deletion of Smad[4] (Smad4fl/fl) and p53 (p53fl/fl) genes to induce lung cancer. Expression of mutant KrasG12D along with p53 and Smad[4] loss-of-function (LOF) engendered a high incidence of metastasis to different tissues, compared to that found in p53fl/fl; KrasG12D mice. Activation of the PAK3-JNK-Jun pathway in the Smad4fl/fl; p53fl/fl; KrasG12D triple-mutant mice contributed to the metastatic potential, suggesting a possible target for therapy of NSCLC. This study provides insights into Smad4-dependent regulation of tumorigenesis, progression, and metastasis in lung cancer

Methods

Results

Conclusion