Cell Death in Hepatocellular Carcinoma: Pathogenesis and Therapeutic Opportunities

Abstract

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer and the third leading cause of cancer death worldwide. Closely associated with liver inflammation and fibrosis, hepatocyte cell death is a common trigger for acute and chronic liver disease arising from different etiologies, including viral hepatitis, alcohol abuse, and fatty liver. In this review, we discuss the contribution of different types of cell death, including apoptosis, necroptosis, pyroptosis, or autophagy, to the progression of liver disease and the development of HCC. Interestingly, inflammasomes have recently emerged as pivotal innate sensors with a highly pathogenic role in various liver diseases. In this regard, an increased inflammatory response would act as a key element promoting a pro-oncogenic microenvironment that may result not only in tumor growth, but also in the formation of a premetastatic niche. Importantly, nonparenchymal hepatic cells, such as liver sinusoidal endothelial cells, hepatic stellate cells, and hepatic macrophages, play an important role in establishing the tumor microenvironment, stimulating tumorigenesis by paracrine communication through cytokines and/or angiocrine factors. Finally, we update the potential therapeutic options to inhibit tumorigenesis, and we propose different mechanisms to consider in the tumor microenvironment field for HCC resolution.

Keywords: nonparenchymal cells; programmed cell death; stromal component; tumor microenvironment.

Figure 1

Programmed cell death pathways initiated by TNF and FAS: (A) The TNF–TNFR1 cascade simultaneously activates proapoptotic and antiapoptotic signals, and diverse signaling complexes are formed. Complex I, composed by the adaptor protein TRADD, the receptor RIPK1, and the E3 ligase TRAF2, enables the recruitment of the inhibitor NF-ƙB kinase complex, comprising IKKα/β and NEMO. Subsequently, the inhibitor of NF-ƙB (IƙBα) is ubiquitinated and NF-ƙB translocates to the nucleus, promoting the expression of survival and antiapoptotic genes such as Bcl2 and c-FLIP. (B) In the FAS/TRAIL-mediated apoptotic pathway, binding of FASL/TRAIL to their cognate receptors results in FAS clustering and binding to FADD. TRADD dissociates from complex I and associates with FADD to form complex IIa, which triggers caspase-8 activation and, consequently, apoptosis. Complex IIb, or ripoptosome, is highly dependent on RIPK1 activity, and represents an alternative death complex that induces apoptosis through direct activation of caspase-8. (C) In contrast, caspase-8 can be inhibited under certain physiological or pharmacological conditions, promoting the shift from the ripoptosome-induced apoptosis to necroptosis, a form of programmed necrosis via complex IIc or necrosome. RIPK3 mediates the phosphorylation of MLKL, resulting in its conformational change and oligomerization. This allows MLKL to bind to plasma membrane lipids and form a pore that leads to cell lysis. Release of cellular components (cytokines, chemokines and DAMPs) perpetuates liver inflammation and fibrogenesis in a pro-tumoral microenvironment, thus promoting HCC development. Abbreviations: Bcl2, B-cell lymphoma 2; Casp-3, caspase-3; c-FLIP, cellular FLICE-like inhibitory protein; DAMPs, damage-associated molecular patterns; FAS, FS-7-associated surface antigen; FADD, FAS-associated death domain protein; FASR, FAS receptor; FASL, FAS ligand; MLKL, mixed lineage kinase domain-like protein; NF-ƙB, nuclear factor kappa-light-chain-enhancer of activated B cells; RIPK1, serine/threonine-protein kinase 1; TNF, tumor necrosis factor; TRADD, TNFRSF1A-associated via death domain; TRAF2, receptor-associated factor; TRAIL, Targeting TNF-related apoptosis-inducing ligand. Created with BioRender.com.

Figure 2

Activation of the NLRP3 inflammasome and canonical pyroptosis in liver cells. Various stimuli of liver damage might cause hepatocyte cell death and gut dysbiosis, leading to high exposure to DAMPs and PAMPs that activate liver inflammasomes: (A) In hepatocytes, NLRP3 inflammasome activation requires two steps: the priming signal (SIGNAL 1) is initiated by PAMPs such as LPS, which bind to their corresponding TLR and upregulate the expression of pro-IL-1β, pro-IL-18, pro-caspase-1, and NLRP3 genes via NF-ƙB signaling activation. The second signal (SIGNAL 2) is triggered by PAMPs and/or DAMPs and activates NLRP3, which, in turn, activates pro-caspase-1. Activated casp-1 cleaves IL-1β and IL-18 precursors into mature and proinflammatory forms that are secreted to extracellular space. Casp-1 and, alternatively, caspase-4/5/11 might also cleave protein GSDMD. The N-terminal fragment of GSDMD (N-GSDMD) forms pores in the plasma membrane, inducing pyroptosis by cell swelling and osmotic lysis. (B) Inflammasome activation and pyroptosis also occurs in nonparenchymal cells. DAMPSs and gut-derived PAMPs activate Kupffer cells via PRRs, triggering the production of IL-1β and, subsequently, CCL2 and TNF. NLRP3 activation in HSC also induces the expression of the profibrogenic molecule TGF-β. Together, these events result in liver inflammation and fibrosis, perpetuating liver damage and hepatocellular carcinoma. Abbreviations: CCL2, C-C motif chemokine ligand 2; DAMPS, damage-associated molecular patterns; GSDMD, gasdermin D; HSCs, hepatic stellate cells; KCs, Kupffer cells; LPS, liposaccharide; NF-ƙc, nuclear factor kappa-light-chain-enhancer of activated B cells NLRP3, NLR family pyrin domain containing 3; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; TLR, toll-like receptor; TNF, tumor-nuclear factor; TGF-β, transforming growth factor beta. Created with BioRender.com.

Figure 3

Myofibroblast-derived cells and matrisome contribution to tumor microenvironment and HCC development: (A) Quiescent HSCs become activated by DAMPs released by apoptotic hepatocytes and by IL-1β and TNFα secreted by polarized M2 macrophages after hepatic injury. aHSCs induce the secretion of proinflammatory growth factors and chemokines such as CCL2, which is recognized by macrophage CCR2, perpetuating liver damage and tumor growth. aHSCs, induced by the loss of hepatocyte FBP1, secrete cytokines such as IL-6 and CXCL1, which are involved in liver tumorigenesis. Growth factor GDF15, induced by aHSC autophagy, also contributes to hepatocarcinoma. (B) CAFs are derived from activated myofibroblasts such as aHSCs. Diverse growth factors and miRNAs contained in EVs and released from cancer cells act as positive stimuli for CAF differentiation. CAFs promote tumor development via the CLCF1–CXCL6/TGFβ signaling pathway and induce the recruitment of macrophages and M2 polarization by endosialin–CD68 interaction. (C) The matrisome plays a key role in hepatocellular carcinoma development by the action of several molecules secreted into the tumor microenvironment, such as MMP, cytokines, integrins, and ADAMs. Abbreviations: aHSCs, activated hepatic stellate cells; qHSCs, quiescent hepatic stellate cells; CAFs, cancer-associated fibroblast cells; CCL2, chemokine (C-C motif) ligand 2; CLCF1, cardiotrophin like cytokine factor 1; ECM, extracellular matrix; EVs, extracellular vesicles; Heps, hepatocytes; HGF, hepatocyte growth factor; HMGB1, high mobility group box protein 1; LSECs, liver sinusoidal endothelial cells; MMP, metalloproteinases; TAMs, tumor-associated macrophages; PDGF, platelet-derived growth factor; TAMs, tumor-associated macrophages; TGF-β, transforming growth factor beta. Created with BioRender.com.

Figure 4

Liver damage activates and dysregulates liver macrophages and LSECs’ role in HCC: (A) KCs are polarized to TAMs, a M2 phenotype macrophage with proangiogenic and proinflammatory properties that act on neighboring cells. Moreover, DAMPs and MAMPs are recognized by PRR on KCs aggravating liver diseases and hepatocellular carcinoma development. Importantly, exhausted KCs are replaced by recruited circulating monocytes, which polarize to TAMs. TAMs can also be recruited through IL-4, IL-10, and CCL2 signals from circulating monocytes. Crosstalk between immune and cancer cells occurs through TGFβ-Tim3 interaction and promotes the polarization of M2 TAMs towards a pro-oncogenic phenotype that triggers tumor growth, invasion, and migration by several secreted factors such as cytokines, proangiogenic factors, and MMPs. (B) Liver damage causes LSECs capillarization, losing specific features such as fenestration and homeostatic proteins (LYVE-1 and STAB1 and 2). Subsequently, LSECs secrete several proinflammatory (IL-8 and CCL2) and adhesion molecules (VCAM-1 and ICAM-1), ECM components (laminin) and growth factors (HGF) that act as angiocrine factors favoring the capillarization process and tumor microenvironment during tumorigenesis. (C) PLVAP+ and VEGFR2+-ECs migrate to tumoral tissue contributing to tumor growth by the formation of new vessels. Moreover, TAEs promote neo-angiogenesis by upregulation of MMP2, inducing ECM remodeling. Abbreviations: CCL2, chemokine (C-C motif) ligand 2; CCR2, C-C chemokine receptor type 2; DAMPS, damage-associated molecular patterns; Heps, hepatocytes; ECs, endothelial cells; ICAM-1, intracellular adhesion molecule 1; KCs, Kupffer cells; LSECs, liver sinusoidal endothelial cells; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1 MAMPs, microbial-associated molecular patterns; PLVAP, plasmalemma vesicle associated protein; PRRs, pattern recognition receptors; STAB, stabilin; TAEs, tumor-associated endothelial cells; TAMs, tumor-associated macrophages cells; VCAM-1, vascular cell adhesion molecule 2. VEGFR2, vascular endothelial growth factor receptor 2. Created with BioRender.com.