MicroRNA-15b in extracellular vesicles from arsenite-treated macrophages promotes the progression of hepatocellular carcinomas by blocking the LATS1-mediated Hippo pathway

Highlights

• Exposure to arsenite, an active form of arsenic, increases miR-15b levels and induces M2 polarization of macrophages.

• Elevated levels of miR-15b are transferred from arsenite-treated macrophages to HCC cells via EVs.

• miR-15b in EVs promotes the progression of HCCs by blocking the LATS1/Hippo pathway.

Abstract

Arsenic, a human carcinogen, causes various human cancers, including those of the skin, lung, and liver. Hepatocellular carcinomas (HCCs), which have high mortality, are common malignancies worldwide. Tumor-associated macrophages (TAMs), which are considered to be similar to M2-polarized macrophages, promote tumor invasion and progression. Small non-coding RNAs (miRNAs) regulate expression of genes involved in progression of various malignancies. Extracellular vesicles (EVs), as mediators of cell communication, pass specific miRNAs directly from TAMs to tumor cells, promoting tumor pathogenesis and metastasis. In HCCs, large tumor suppressor kinase 1 (LATS1), functions as a tumor suppressor. However, the molecular mechanism by which miRNA modulates LATS1 expression in HCCs remains unclear. The results show that exposure to arsenite, increased miR-15b levels and induced M2 polarization of THP-1 cells. Elevated levels of miR-15b were transferred from arsenite-treated-THP-1 (As-THP-1) cells to HCC cells via miR-15b in EVs inhibited activation of the Hippo pathway by targeting LATS1, and was involved in promoting the proliferation, migration, and invasion of HCC cells. In conclusion, miR-15b in EVs from As-THP-1 cells is transferred to HCC cells, in which it targets and downregulates LATS1 expression and promotes the proliferation, migration, and invasion of HCC cells.

Introduction

Arsenic is an environmental toxicant and human carcinogen that may be present in soil, air, food, and water [1]. Most cases of arseniasis are caused by drinking groundwater contaminated with arsenic [2]. In some countries, such as Bangladesh, India, China, Chile, and Mexico, the arsenic content in drinking water in some areas is much higher than “Drinking Water Hygiene Standard”, which stipulates that the content of arsenic in drinking water shall not exceed 0.01 mg/L and that the maximum allowable concentration is 0.05 mg/L [3,4]. The liver is the main organ for arsenic storage and metabolism [5], and accumulation of arsenic in the liver relates to arsenic exposure. Chronic, low-dose exposure to arsenic causes a variety of human cancers, including those of the skin, lung, and liver [6,7]. Arsenic causes liver damage, fibrosis, cirrhosis, and hepatocellular carcinomas (HCCs) through genomic instability, changes in signaling pathways, oxidative stress, DNA methylation, and non-coding RNAs [8]. However, the molecular mechanisms of how arsenic exposure causes these diseases remain unclear.

HCCs, the main histological subtype of liver cancer, have a high mortality rate and account for 90% of primary liver cancers [9]. HCCs are common malignancies, often induced by chronic exposure to arsenic [10]. The incidences of liver cancer and other liver-related diseases for people with arsenic poisoning are higher than those for healthy people [11,12]. Our previous results reveal that chronic exposure to arsenite causes malignant transformation of normal liver cells (L-02 cells) [13,14]. Despite advances in early diagnosis of HCCs and specialized treatments for HCC, the prognosis for these patients remains poor due to high rates of recurrence and metastasis [15,16]. Therefore, it is important to understand the pathophysiological conditions that contribute to HCC carcinogenesis and progression, in order to seek new diagnostic and therapeutic strategies and thereby to improve the overall prognosis for HCC patients.

Macrophages, which are widely distributed immune cells, recognize, engulf, and eliminate dead cells and pathogens [17,18]. In general, macrophages are polarized to a classically activated (M1) or alternative activated (M2) state by responding to various stimuli [19]. M1 macrophages are thought to release pro-inflammatory and immunostimulatory cytokines to inhibit tumor occurrence and development [20]. M2 macrophages are thought to have similar functions to tumor-associated macrophages (TAMs) [21]. TAMs promote tumor cell proliferation, invasion, and metastasis; stimulate tumor-related angiogenesis; and inhibit the anti-tumor immune response [22,23]. Because TAMs are involved in promoting tumor formation and metastasis, they are recognized as potential targets for the treatment of cancers [24]. Clinical and experimental evidence shows that TAMs promote HCCs initiation and progression [25]. However, the underlying molecular mechanisms by which TAMs promote cancer metastasis and communication between TAMs and tumor cells are poorly understood.

Extracellular vesicles (EVs) are lipid bilayer vesicles (50–100 nm) of endocytic origin, which mediators of cellular communication that are involved in the progression of tumors [26,27]. When multi-vesicular bodies fuse with cell membranes, EVs are released into the extracellular environment [28]. EVs mediate communication between tumor cells and microenvironmental cells and participate in various physiological and pathological processes [29,30]. For example, transglutaminase-2 facilitates EVs-mediated establishment of metastases [31]. HCCs-derived miRNA-21 in EVs contributes to tumor progression by converting hepatocyte stellate cells to cancer-associated fibroblasts [32]. EVs derived from hypoxic epithelial ovarian cancer cells deliver microRNA-940 to induce macrophage M2 polarization [33]. Thus, EVs promote tumor growth, tumorigenesis, tumor angiogenesis, tumor immune escape, drug resistance, and metastasis.

The Hippo pathway is involved in maintaining the size of the liver and in inhibiting the occurrence and metastasis of HCCs [34,35]. It consists of a series of conserved kinases, which control organ size and volume by regulating cell proliferation and apoptosis [36]. When a cell receives a growth inhibitory signal, it activates a series of kinase cascade phosphorylation reactions, and the activated LATS1/2 kinase phosphorylates downstream effectors, YAP and TAZ. A cytoskeleton protein, 14-3-3, binds to phosphorylated YAP and TAZ, which keeps them in the cytoplasm and prevents their nuclear transcription. Conversely, unphosphorylated YAP/TAZ enters the nucleus, binds to TEAD1-4 or other transcription factors, and induces expression of apoptosis-inhibiting genes [37]. In summary, with exposure to arsenite, Hippo signaling is inhibited, and YAP and TAZ are activated and transferred to the nucleus, thereby promoting tumor cell proliferation.

In the present work, we evaluated the effect of miR-15b in EVs from arsenite-treated-THP-1 (As-THP-1) cells on the malignancy of HCC cells and investigated the underlying mechanisms. First, arsenite exposure induced THP-1-derived macrophages to polarize to M2 macrophages. Then, HCC cells were treated with the culture medium, and EVs were prepared from the As-THP-1 cells. The results demonstrated that miR-15b, transferred from As-THP-1 cells into HCC cells via EVs, functioned as an extracellular signaling molecule that inhibited the Hippo pathway and promoted cell proliferation, migration, and invasion. The down-regulation of large tumor suppressor kinase 1 (LATS1) in HCC cells was a response to miR-15b in EVs.

In sum, our findings unveil a new connection between As-THP-1 cells, EVs, and HCC cells: miR-15b is shuttled from As-THP-1 cells via EVs to HCC cells, where it downregulates LATS1 expression, which in turn inhibits Hippo pathways and promotes the progression of HCCs. The present study explores the molecular regulatory mechanisms of arsenite in promoting the formation and progression of HCCs. Such information is essential for the prevention and treatment of arsenic damage.