Recently, Jian Liu’s group from Zhejiang University-University of Edinburgh Institute (ZJE) in Zhejiang University published their lung cancer metastasis research entitled “Loss of Smad4 promotes aggressive lung cancer metastasis by de-repression of PAK3 via miRNA regulation” at Nature Communications by collaborating with multiple groups, including the Pro. Xiao Yang from State Key Laboratory of Proteomics in National Center for Protein Sciences (Beijing) and Pro. Xiaotao Li from Shanghai Key Laboratory of Regulatory Biology in East China Normal University, and Pro. Dianwen Song from The First People’s Hospital of Shanghai Jiao Tong University. By developing different genetic mouse models mimicking lung cancer metastasis and the comprehensive mechanism studies, they revealed that the de novo role of SMAD4 in repressing lung cancer metastasis, independent of TGFβ, and the therapeutic targets (SMAD4/miRNA-495/PAK3) for treating lung cancer metastasis.
Access to the publication：https://www.nature.com/articles/s41467-021-24898-9
Lung cancer is the leading cause of cancer death worldwide and in China1. Over 85% of lung cancer patients harbor overt or subclinical metastases at diagnosis, and therefore most patients die of progressive metastatic disease despite aggressive local and systemic therapies1. SMAD4 is the crucial downstream transcription factor of the TGFβ pathway and has been found to have some somatic mutations and the decreased expression in human lung tumors2,3. Although Jian Liu et al. reported that Smad4 ablation promoted lung cancer development and metastasis, the mechanism of SMAD4 in regulating lung cancer metastasis remains unclear.
Jian Liu et al. have reported that ablation of Smad4 in mouse lungs could not cause cell hyperplasia and tumor formation2,6. This research found that some lung cancer patients harbored the mutations of KRAS, TP53, and SMAD4 in the same tumors by analyzing the TCGA lung cancer datasets. Therefore, this research generated KrasG12DTrp53flox/flox (PK) and KrasG12DTrp53flox/floxSmad4flox/flox (SPK) mouse models to mimic the lung cancer metastasis to investigate the role of SMAD4 in regulating lung cancer under the background of Kras mutation and Trp53 ablation. Moreover, this research found that the ratio of lung tumor metastasis in SPK mice was much higher than that of PK mice. By conducting the transcriptome analysis of the isolated lung tumor cells from PK and SPK mice, followed by the functional analyses (e.g., cell invasion), this research found that Smad4 ablation increased the mRNA and protein expression of PAK3 (Figure 1, Step 3), and consequently activated the JNK-JUN pathway of the downstream of PAK3 (Figure 1, Steps 4 and 5), and eventually promoted lung cancer metastasis. Unexpectedly, the negative regulation of SMAD4 on PAK3 is independent of TGFβ. Analysis of SMAD4 ChIP-Seq data showed that there was no SMAD4 binding on the PAK3 promoter.
Furthermore, the exogenous expression of SMAD4 did not alter the luciferase activity driven by the PAK3 promoter. Thus, it indicated that the negative regulation of SMAD4 on PAK3 is independent of TGFβ, belonging to the non-classic role of SMAD4. Some published studies showed that the transcriptional regulation of gene expression could be regulated either by the promoter or the 3’ UTR7. This research further showed that Smad4 loss altered the luciferase activity driven by the PAK3 3’ UTR. Given that miRNA can negatively regulate gene expression by binding on the targeting gene’s 3’ UTR, this research raised a hypothesis that SMAD4 negatively regulates the PAK3 mRNA expression through miRNAs. Through the bioinformatical analysis, as well as the functional validations, this research found that miR-495 and miR-543, directly and positively regulated by SMAD4, mediated the negative regulation of SMAD4 on PAK3 (Figure 1, Steps 1 and 2). Clinically, the negative correlation between SMAD4 and PAK3-JNK-JUN was observed in a group of human lung cancer samples, including the control lungs, early-stage lung tumors, late-stage lung tumors, and metastatic lung tumors. In sum, this research identified SMAD4/miRNA-495/PAK3 to be the de novo targets in treating lung tumor metastases.
Figure 1. The TGFβ-independent role of SMAD4 in regulating lung cancer metastasis
Of note, the SMAD4 repression on PAK3 is independent of TGFβ since SMAD4 is a typical functional player of the TGFβ pathway. Moreover, they showed that miRNA-495 was able to prevent the lung cancer metastasis induced by Smad4 loss. The biggest challenge of the application of RNA drugs is the in vivo stability of RNA8,9. Given that the mRNA vaccine (Moderna) was successful against COVID-19, it suggested that the concerns of the low stability of RNA drug in vivo have been partially resolved during the clinical applications. Therefore, miRNA-495 against lung cancer metastasis may benefit developing clinical treatment.
Dr. Jian Liu is the co-corresponding author of the article. In addition, the master student Feiran Gao (ZJE, 2020 entry) participates in this research. To overcome the difficulties of this research, such as a large number of genetic mouse experiments, the non-classic role of SMAD4, and the different stages of human lung cancer samples, all collaborators effectively communicated with each other and worked together. For example, Dr. Xiao Yang’s group was mainly responsible for generating the mouse models; Drs. Xiaotao Li and Lei Li’s group was mainly in charge of the mechanism investigation and the initial version of the manuscript; Dr. Jian Liu’s group took the responsibility to conduct all bioinformatic analyses, parts of experimental designs, and the whole revision process; Drs. Dianwen Song’s and Junjiang Fu’s groups collected the clinical samples. Meanwhile, the international collaborators also contributed to this research, including Dr. Nabeel Bardeesy from Massachusetts General Hospital of Harvard University, Dr. Kwok-kin Wong from Laura and Isaac Perlmutter Cancer Center of New York University, and Dr. Robb E. Moses from Dan L. Duncan Cancer Center of Baylor College of Medicine.
About Jian Liu’s lab (https://person.zju.edu.cn/en/jian_liu)
References
1. Rebecca L Siegel, et al. Cancer statistics, 2019. CA Cancer J Clin (2019).
2. Xiaohong Tan, et al. Loss of Smad4 promotes aggressive lung cancer metastasis by de-repression of PAK3 via miRNA regulation. Nature Communications (2021)
3. Jian Liu. et al. ErbB2 Pathway Activation upon Smad4 Loss Promotes Lung Tumor Growth and Metastasis. Cell Reports (2015).
4. Jian Liu, et al. ERBB2 Regulates MED24 during Cancer Progression in Mice with Pten and Smad4 Deletion in the Pulmonary Epithelium. Cells (2019).
5. Ran You, et al. IL17A regulates tumor latency and metastasis in lung adeno and squamous SQ. 2b and AD. 1 cancer. Cancer immunology research (2018)
6. Jian Liu. et al. JNK1/2 represses Lkb1-deficiency-induced lung squamous cell carcinoma progression. Nature Communications (2019)
7. Yuval Malka. et al. Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments. Nature Communications (2017)
8. Ugur Sahin. et al. mRNA-based therapeutics — developing a new class of drugs. Nature Reviews Drug Discovery (2014)
9. Meirav Segal. Et al. Challenges identifying efficacious miRNA therapeutics for cancer. Expert Opinion on Drug Discovery (2020)
