TLR3 induces Hepatocellular carcinoma cell death and increases natural killer cell activity

Abstract

Hepatocellular Carcinoma Cancer (HCC) is a highly aggressive cancer generally caused by chronic liver disease. A persistent, non-specific and ineffective activation of the immune system within the chronically infected liver has been proposed as the main cause of repeated cycles of tissue damage, repair and regeneration that eventually promote carcinogenesis. However, appropriate activation of the immune system can also prevent tumour progression. We hypothesized that the tumour immune microenvironment could affect the progression of established tumours and evaluated the potential immune mechanisms associated with clinical end point in HCC. Our results show that HCC patient survival is positively correlated with a proinflammatory innate immune gene signature within the tumours. This signature includes cytokines such as TNF/ IL-6 and LTA and two Toll-Like-Receptors(TLR): TLR3 and TLR4. Importantly, the expression of TLR3 is found to be positively associated with patient survival in two independent cohorts of patients. In addition, the survival-associated cytokines are mostly derived from tumour-infiltrating immune cells while TLR3 is expressed by both immune and tumour cells. We show that some HCC cell lines express TLR3 and stimulation of these cell lines with polyinosinic-polycytidilic acid (Poly I:C), a TLR3 ligand, results in an increase in TLR3 expression and subsequent tumour cell death by 24 hr. Proliferating immune cells, predominantly NK and T cells are also found in tumours of patients with longer survival. The density of these cells is correlated positively with cancer cell apoptosis and negatively with their proliferation. Our data identify TLR3-expressing leukocytes to be NK cells. NK cells express TLR3 and can upregulate TLR3 upon poly I:C stimulation. Furthermore NK cells, even in the absence of antigen-presenting cells, upregulate IFN-? secretion in response to poly I:C. Data from our lab showed that IFN-? potentiates CXCL10 secretion by HCC cells and macrophages and that CXCL10 is attracting Th1 and NK cells. Therefore our finding suggest that poly I:C-induced expression of IFN-? by NK cells can reinforce this paracrine loop, leading to increased activity towards tumours. NK cells isolated from PBMC of normal donors and treated with poly I:C showed increased expression of TLR3, granzyme B and perforin. They also display an increased killing of HCC cells. Finally, TLR3 expression decreases with the advancement of the disease in HCC patients. Patients with lower grade and early stages of HCC have higher TLR3 expression than patients with higher grades and more advanced stages. This suggests that low expression of TLR3 by cancer cells confers a selective advantage for the tumour, since TLR3 can result in cell death of the tumour, either directly or through the action of immune cells such as NK cells. Therefore, an inflammatory environment within the HCC tumour could be an important means to control tumour progression via immune cells activation and proliferation. Such activation may be promoted by danger signals sensed by TLR such as TLR3 on both NK and tumours.