Summary
Liver metastasis is a major cause of death in patients with colorectal cancer (CRC). Fatty liver promotes liver metastasis, but the underlying mechanism remains unclear. We demonstrated that hepatocyte-derived extracellular vesicles (EVs) in fatty liver enhanced the progression of CRC liver metastasis by promoting oncogenic Yes-associated protein (YAP) signaling and an immunosuppressive microenvironment. Fatty liver upregulated Rab27a expression, which facilitated EV production from hepatocytes. In the liver, these EVs transferred YAP signaling-regulating microRNAs to cancer cells to augment YAP activity by suppressing LATS2. Increased YAP activity in CRC liver metastasis with fatty liver promoted cancer cell growth and an immunosuppressive microenvironment by M2 macrophage infiltration through CYR61 production. Patients with CRC liver metastasis and fatty liver had elevated nuclear YAP expression, CYR61 expression, and M2 macrophage infiltration. Our data indicate that fatty liver-induced EV-microRNAs, YAP signaling, and an immunosuppressive microenvironment promote the growth of CRC liver metastasis.
Introduction
Colorectal cancer (CRC) is the third most common malignancy and the second leading cause of cancer-related death, accounting for about 900,000 deaths per year worldwide.1 The liver is the most common metastatic site for CRC, owing to the unique anatomical link between the intestine and the liver through the portal vein. The liver-specific metabolic and immune microenvironment also supports CRC liver metastasis. Ultimately, 70% of patients with CRC will develop liver metastasis, which is the major cause of death.2,3
Obesity and non-alcoholic fatty liver disease (NAFLD) are the significant risk factors for CRC.4 More than 650 million adults are obese worldwide, and the increased prevalence of NAFLD is linked to the obesity epidemic. A growing body of epidemiological evidence indicates that fatty liver increases the occurrence of CRC liver metastasis and the local recurrence after resection of CRC liver metastases, thereby worsening prognosis.5,6,7,8,9,10,11,12,13,14,15 Likewise, fatty liver augments metastatic liver tumor growth in animal models of CRC liver metastasis by altering the hepatic inflammatory and immune microenvironment.16 These findings suggest metastatic mechanisms may differ owing to heterogeneity of the tumor microenvironment (TME) in fatty liver, which may explain diverse responses to cancer therapies among patients. As such, disease management for liver metastasis may differ in patients with or without fatty liver. There is an urgent need to understand the molecular mechanisms of metastasis in patients with fatty liver to manage those patients effectively.
Extracellular vesicles (EVs) contain bioactive macromolecules, such as microRNAs (miRNAs), lipids, and proteins. EV production and secretion into the extracellular space is regulated by ceramide synthesis via neutral sphingomyelinase and Rab proteins such as Rab27a and Rab27b.17,18 Rab27a mediates EV production in melanoma and its metastasis, and is important for EV secretion enhanced by lipid overload.19,20,21 In NAFLD, hepatocyte-derived EV production is increased along with altered miRNA contents.22 Previous studies showed that primary tumor-derived EVs travel to the liver and facilitate pre-metastatic niche formation and pro-metastatic inflammatory responses.23,24,25,26,27 However, the contribution of EVs produced in the liver, particularly under NAFLD conditions, toward the formation of a pre- and pro-metastatic liver environment that predisposes to CRC liver metastasis has not been explored.
Yes-associated protein (YAP), a transcriptional co-regulator, is the effector of the Hippo pathway, which regulates the activity of pro-cancerous transcription factors, such as transcriptional enhanced associate domains (TEADs).28 When YAP is phosphorylated by the mammalian Ste20-like kinases 1/2 (MST1/2) and by the large tumor suppressor kinases 1/2 (LATS1/2), YAP is localized in cytoplasm and inhibits its activity by proteasomal degradation.29 In contrast, unphosphorylated YAP is localized to the nucleus and activates TEAD transcription factors, promoting gene expression for cancer growth, invasion, and metastasis.29 G protein-coupled receptors, mechanical cues, cell adhesion, and integrin signaling regulate YAP activity in a phosphorylation-dependent manner, but YAP activity is also regulated post-transcriptionally by miRNAs.30,31 Pro-cancerous YAP activity intrinsically contributes to cancer cell behavior, but it also affects surrounding immune cells. Loss of LATS1/2 kinases enhances anti-cancer immunity,32 but high YAP activity induces an immunosuppressive microenvironment by recruiting M2 tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells or by upregulating programmed cell death 1 ligand (PD-L1) expression.33,34,35,36,37
In the present study, we reveal that fatty liver produces EVs that contain pro-carcinogenic miRNAs and creates a pre-metastatic and pro-metastatic liver microenvironment that predisposes to CRC liver metastasis. Our results indicate that EV transfer of pro-carcinogenic miRNAs from fatty liver hepatocytes to metastatic cancer cells increases YAP activity by inhibiting LATS2. We show that heightened YAP activity promotes CRC liver metastatic growth by enhancing M2-TAM recruitment and CD8 T cell exhaustion to create an immunosuppressive microenvironment. Thus, NAFLD likely generates a complex, metastatic TME, contributing to CRC liver metastasis. Further, the recent increase in global burden of NAFLD may explain the diverse responses to cancer therapies for patients with CRC and liver metastasis.
Section snippets
Increased extracellular vesicle release by fatty liver enhances metastatic tumor growth in the liver
To investigate whether fatty liver affects metastatic CRC growth in the liver, wild-type (WT) mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 6 weeks followed by splenic injection of syngeneic MC38 CRC cells for an additional 2 weeks to establish a CRC liver metastasis model with or without fatty liver (Figure 1A). The HFD caused an increase in the number and size of metastatic tumors along with increased serum EV particles (Figures 1A–1C). The distribution of EV particle sizes
Discussion
Recent evidence has demonstrated that NAFLD is more tightly associated with a risk of primary cancers of liver, breast, prostate, pancreas, and colorectum than with a risk of obesity alone.4 As 25% of adults in the United States have NAFLD and its prevalence is increasing,54 NAFLD-associated cancers are a significant health issue. The incidence and recurrence rates for CRC liver metastasis are higher in patients with fatty liver than in patients with non-fatty liver….
