Cancer-associated fibroblasts activated by miR-196a promote the migration and invasion of lung cancer cells

doi:10.1016/j.canlet.2021.03.021

Cancer Letters

Volume 508, 28 June 2021, Pages 92-103

https://doi.org/10.1016/j.canlet.2021.03.021 Get rights and content

Highlights

•
CAF-specific miR-196a promotes the migration and invasion of lung cancer cells.
•
The effect of miR-196a is medicated by ANXA1 and CCL2.
•
miR-196a is associated with poor survival of lung adenocarcinoma patients.
•
miR-196a will be a good therapeutic target in lung adenocarcinomas.

Abstract

Fibroblasts in the tumor microenvironment, known as cancer-associated fibroblasts (CAFs), promote the migration, invasion, and metastasis of cancer cells when they are activated through diverse processes, including post-transcriptional regulation by microRNAs (miRNAs). To identify the miRNAs that regulate CAF activation, we used NanoString to profile miRNA expression within normal mouse lung fibroblasts (LFs) and CAFs. Based on NanoString profiling, miR-196a was selected as a candidate that was up-regulated in CAFs. miR-196a-overexpressed LFs (LF-196a) promoted the migration and invasion of lung cancer cells in co-culture systems (Transwell migration and spheroid invasion assays). ANXA1 was confirmed as a direct target of miR-196a, and adding back ANXA1 to LF-196a restored the cancer cell invasion promoted by miR-196a. miR-196a increased CCL2 secretion in fibroblasts, and that was suppressed by ANXA1. Furthermore, blocking CCL2 impeded cancer spheroid invasion. In lung adenocarcinoma patients, high miR-196a expression was associated with poor prognosis. Collectively, our results suggest that CAF-specific miR-196a promotes lung cancer progression in the tumor microenvironment via ANXA1 and CCL2 and that miR-196a will be a good therapeutic target or biomarker in lung adenocarcinoma.

Introduction

Lung cancer is a major cause of cancer-related death worldwide [1]. Although diverse therapies against lung cancer have been developed, the 5-year survival rate of lung cancer patients is still much lower than that of other cancer patients [2]. The poor prognosis of lung cancer patients is mostly due to the high prevalence of metastasis [3]. The metastatic potential of cancer cells is determined by their direct or indirect interactions with the multiple types of surrounding cells, such as immune cells, blood vessel cells, and fibroblasts, that compose the tumor microenvironment along with secretory signaling factors and the extracellular matrix. The tumor microenvironment associated with lung cancer progression has been attracting attention in investigations of effective prognostic and therapeutic targets [4]. To achieve effective patient treatment and improve patient survival rates, novel anti-cancer strategies targeting both cancer cells and the tumor microenvironment are urgently required.

Fibroblasts in the tumor microenvironment promote the proliferation, epithelial-mesenchymal transition (EMT), migration, and invasion of cancer cells. Cancer-associated fibroblasts (CAFs) promote EMT and tumor progression by secreting cytokines and growth factors such as IL-6, VEGF-A, EGF, and TGF-β in lung cancer [5,6] and endometrial cancer [7]. Moreover, CAFs regulate cancer invasion through direct physical interactions with cancer cells [8]. This direct interaction enhances cancer stemness in pancreas cancer [9]. Through both paracrine signaling and physical contact, CAFs facilitate cancer development and metastasis.

The influence of CAFs on cancer exacerbation can be modulated by microRNAs (miRNAs). miRNAs are endogenous inhibitors of gene expression that silence target mRNAs by inducing mRNA cleavage or blocking protein translation [10]. As a form of paracrine signaling, certain miRNAs packaged in exosomes are released externally and affect the physiological activities of neighboring cells. In the tumor microenvironment, CAFs enhance cancer cell proliferation, drug resistance, and metastasis via exosome-mediated miRNA delivery [11,12]. Cancer cells also stimulate stromal fibroblasts by providing their miRNAs via exosomes in the tumor microenvironment [13].

Obviously, intracellular miRNAs can also modulate the functions of CAFs in the tumor microenvironment. During the process of CAF activation, global miRNA expression changes occur; however, the functions and roles of CAF-specific miRNAs have not yet been studied in detail. Therefore, in this study, we sought to identify CAF-specific miRNAs and demonstrate their roles in the tumor microenvironment. We performed miRNA expression profiling in CAFs and normal lung fibroblasts (LFs) using the NanoString method and identified numerous miRNAs that were up- or down-regulated in CAFs compared with LFs. Among them, we selected miR-196a, which is up-regulated in CAFs, for further study. CAFs with high miR-196a expression enhanced cancer cell migration, invasion, and metastasis. In lung adenocarcinoma patients, high miR-196a expression was found to be associated with poor prognosis. These findings suggest that CAF-specific miR-196a can be considered as a cancer biomarker and a new therapeutic target in lung adenocarcinoma.

Section snippets

Cell culture

344SQ, 344LN, 531LN2 (murine) and A549 (human) lung cancer cells were cultured in RPMI 1640 (Welgene, Gyeongsan, Korea) with 10% fetal bovine serum (FBS; HyClone, Logan, UT) at 37 °C in the presence of 5% CO₂. To authenticate A549 cell line, short tandem repeat profiling was performed using Powerplex 18D kit (Promega, Madison, WI). Normal murine LFs were isolated from the lungs of 129/Sv wild type mice, and CAFs were isolated from the lung tumors of Kras-mutant (Kras^LA1) mice using

CAFs promote migratory and invasive activity of lung cancer cells

To investigate the effects of CAF-specific miRNAs on the activity of lung cancer cells, we first isolated murine LFs and CAFs using magnetic-activated cell sorting. LFs were isolated from the lungs of 129/Sv wild type mice, and CAFs were isolated from the lung tumors of Kras-mutant (Kras^LA1) mice [6] using THY-1, a fibroblast-specific marker [16]. The isolated LFs and CAFs showed morphological differences in 2-D cultures. To observe the morphology more clearly, actin cytoskeletons of LFs and

Discussion

In tumor microenvironments, stromal CAFs and cancer cells interact through diverse direct and indirect mechanisms in which miRNAs can function as key mediators [37,38]. miRNAs can directly regulate CAFs’ typical features by intervening in differentiation and reprogramming processes. In this work, we used spheroid 3-D cultures composed of lung cancer cells and CAFs to show that miR-196a induces the reprogramming of stromal fibroblasts in ways that facilitate cancer cell invasion. We also

Authors’ contributions

SL, JHH, JSK, JSY, SHC, EJK, JLK and Y-HA performed the experiments. SL, YHK and Y-HA designed the study. SL, JHH, SHC, SAH, KK and Y-HA analyzed the data. SL and Y-HA wrote the manuscript.

Declaration of competing interest

The authors declare no potential conflicts of interest.

Acknowledgements

The authors thank Dr. Jonathan M. Kurie (University of Texas MD Anderson Cancer Center) for his valuable suggestions. This research was supported by grants of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI17C0666 to Y-HA; HI20C0461 to SL), and by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (NRF-2020R1A5A2019210 to Y-HA; NRF-

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Cancer-associated fibroblasts activated by miR-196a promote the migration and invasion of lung cancer cells

Highlights

Abstract

Introduction

Section snippets

Cell culture

CAFs promote migratory and invasive activity of lung cancer cells

Discussion

Authors’ contributions

Declaration of competing interest

Acknowledgements

Mol. Canc.

Trends Canc.

Biochem. Biophys. Res. Commun.

Trends Mol. Med.

Eur. J. Canc.

J. Biol. Chem.

J. Bone Oncol.

Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries

Ca - Cancer J. Clin.

Cancer treatment and survivorship statistics

Ca - Cancer J. Clin.

Prognosis and predictors of site of first metastasis after definitive radiation therapy for non-small cell lung cancer

Acta Oncol.

The lung microenvironment: an important regulator of tumour growth and metastasis

Nat. Rev. Canc.

Cancer-associated fibroblasts enhance metastatic potential of lung cancer cells through IL-6/STAT3 signaling pathway

Oncotarget

miR-200 Inhibits lung adenocarcinoma cell invasion and metastasis by targeting Flt1/VEGFR1

Mol. Canc. Res.

Cancer-associated fibroblasts induce epithelial-mesenchymal transition through secreted cytokines in endometrial cancer cells

Oncol. Lett.

Direct interaction between carcinoma cells and cancer associated fibroblasts for the regulation of cancer invasion

Cancers

Direct interactions with cancer-associated fibroblasts lead to enhanced pancreatic cancer stem cell function

Pancreas

MicroRNA (miRNA) in cancer

Canc. Cell Int.

Exosomal miR-196a derived from cancer-associated fibroblasts confers cisplatin resistance in head and neck cancer through targeting CDKN1B and ING5

Genome Biol.

Inhibition of microRNA-196a might reverse cisplatin resistance of A549/DDP non-small-cell lung cancer cell line

Tumour Biol.

Tumour-originated exosomal miR-155 triggers cancer-associated cachexia to promote tumour progression

Mol. Canc.

Contextual extracellular cues promote tumor cell EMT and metastasis by regulating miR-200 family expression

Genes Dev.

Octopus-toolkit: a workflow to automate mining of public epigenomic and transcriptomic next-generation sequencing data

Nucleic Acids Res.

Thy-1(+) cancer-associated fibroblasts adversely impact lung cancer prognosis

Sci. Rep.

Cancer-associated fibroblasts: their characteristics and their roles in tumor growth

Cancers

CXCR2 expression in tumor cells is a poor prognostic factor and promotes invasion and metastasis in lung adenocarcinoma

Cancer Res.

MicroRNA-196a promotes non-small cell lung cancer cell proliferation and invasion through targeting HOXA5

BMC Canc.

dbDEMC 2.0: updated database of differentially expressed miRNAs in human cancers

Nucleic Acids Res.

Cancer-associated fibroblasts regulate the plasticity of lung cancer stemness via paracrine signalling

Nat. Commun.

Dysregulation of mir-196b in head and neck cancers leads to pleiotropic effects in the tumor cells and surrounding stromal fibroblasts

Sci. Rep.

Genetic polymorphism of miR-196a-2 is associated with bone mineral density (BMD)

Int. J. Mol. Sci.

ANXA1 inhibits miRNA-196a in a negative feedback loop through NF-kB and c-Myc to reduce breast cancer proliferation

Oncotarget

miR-196a regulates the proliferation, invasion and migration of esophageal squamous carcinoma cells by targeting ANXA1

Oncol. Lett.