Key words : Apoptosis, Cell death, Cell switch, Cholangiocarcinoma, Hepatocellular carcinoma

Introduction

The precise balance between proliferation and cell death is critical for the survival of any multicellular organism. Cell death fundamentally impacts the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation, and immune surveillance.^1,2 Regular cell death plays a critical role in regulating the fate of individual cells during adult life and preventing many human diseases, especially tumor formation. Multiple mechanisms of regular cell death have been identified that function in distinct manners. Apoptosis and autophagy are programmed cell death, whereas necrosis is considered an accidental, unprogrammed, or unregulated cell death that occurs upon cellular insults by physical stresses.^1-6

In recent years, extensive research has uncovered novel cell death pathways independent of apoptosis. New interest in necrosis has been sparked by the recent discovery of different forms of necrosis that show regulated pathways. These types of regular cell deaths have been examined under the term “regulated necrosis” and are characterized by cellular swelling, cytoplasmic granulation, and cell membrane leakage with the release of cellular components into the surrounding.7^,8 Various forms of regular necrosis regulated by various molecular pathways have been recently defined, such as necroptosis, parthanatos, oxytosis, ferroptosis, pyronecrosis, and pyroptosis.^9,10 Among these, necroptosis, which has features mimicking apoptosis and necrosis, is a regulated, inflammatory form of cellular death. Necroptosis is considered a backup pathway in cases where apoptosis cannot occur.^6-10

Necroptosis has been associated with many diseases, such as cancer and autoimmune, neu-rodegenerative, and inflammatory diseases.^1,2,8 Numerous human molecules have been shown to play a role in regulation of necroptosis. Both caspase-dependent and caspase-independent mechanisms control the pathway of necroptosis. Mixed lineage kinase domain-like pseudokinase (MLKL), receptor-interacting serine/threonine-protein kinase 1 (RIPK1), and receptor-interacting serine/threonine-protein kinase 3 (RIPK3) are the critical molecules in the regulation of necroptosis.^2,6,11-15 There are more than 20 drugs that can act on molecules involved in necroptosis regulation.

Activation of RIPK1 and RIPK3 by phospho-rylation leads to necrosome formation.^2,6,11,12 The formation of the necrosome is a critical event in necroptosis activation. The interaction of RIPK1 and RIPK3 and the formation of the necrosome induce phosphorylation of MLKL.^11-14 The binding of MLKL to the membrane causes its rupture and release of damage-associated molecular patterns (DAMPs). Damage-associated molecular patterns bind to the cell surface receptors of innate immune cells, leading to increased transcription of proinflammatory cytokines and increased inflammation.^2,6,15,16

In association with DAMP release, there are divergent modes of cell death. Typically, apoptosis minimizes inflammation through sequestration and inactivation of intracellular DAMPs due to early recognition and removal of apoptotic cells via local phagocytes. Necrotic cell death promotes the liberation of intracellular DAMPs to promote inflammation. Certain stimuli (tumor necrosis factor α, Toll-like receptor 3 engagement) that generally result in caspase-dependent apoptosis can be shifted to necroptosis via inhibition of caspase activation, resulting in DAMP release.^2,6,15,16

Necroptosis-associated inflammation through activation of DAMPs is suggested to have both beneficial or adverse effects in cancer.^6,17-19 Cells undergoing necroptosis have been shown to release DAMPs, activating both innate and adaptive immunity. Through the release of DAMPs into the tumor microenvironment, necroptotic cells may provide both antigens and specific cytokines such as interleukin 1a (IL-1a). Interleukin 1a activates dendritic cells to produce IL-12, a cytokine that is critical for the anticancer response.²⁰ Dendritic cells stimulated by inflammatory DAMPs also induce the cross-priming of CD8-positive effector T cells to develop effective antitumoral immunity.^6,21,22 Consequently, DAMPs have a favorable influence on anticancer treatment once immunogenic necroptosis is induced. However, it should be noted that both the secretion of IL-1a and the activation of dendritic cells are strictly dependent on RIPK3 expression in tumor cells.^21,22

Necroptosis and Primary Liver Cancer Subtypes
Patients with primary liver carcinoma have high cancer-related mortality, and the treatment options are limited, especially in those at advanced stages. Liver cancer is a heterogeneous group of tumors that include mostly the epithelial tumors. As with other malignant tumors, tumor development results from an accumulation of genetic and epigenetic alterations.^23-25

Cirrhosis and chronic liver inflammation represent the most critical risk factors for the development of primary liver cancer. Hepatocellular carcinoma (HCC) and intrahepatic cholangiocar-cinoma (ICC) are the most common primary liver tumors, each showing distinct morphology, progression, and metastatic capacity. Hepatocellular carcinoma is mainly restricted to the liver and shows a local invasive growth, whereas ICC tends to metastasize to distant organs. Based on general knowledge, primary liver cancer is considered to develop from hepatocytes, cholangiocytes, hepatoblasts, and liver stem/progenitor cells.^23-25 Hence, it was previously believed that HCC originated from hepatocytes and ICC from cholangiocytes. However, sometimes the same type of cell may give rise to reasonably different types of cancer, which can show significant differences in histopathology, progression, and prognosis.

Recent studies have confirmed this suggestion by showing that HCC and ICC may originate from mature hepatocytes rather than liver progenitor cells, hepatic stellate cells, and cholangiocytes.^26-30 The reason why hepatocytes can develop different types of cancer during chronic liver injury is thought to be because they have a high degree of cellular plasticity. However, the underlying mechanism that determines the phenotype of the tumor cell during liver cancer development is not yet fully known. It has been suggested that the features of the tumor microenvironment, such as an inflammatory microenvironment caused by viral hepatitis or steatohepatitis, and necroptosis-associated hepatic cytokine microenvironment are critical in the determination of tumor phenotype during the development of primary liver cancer.^27-29 Recently, Seehawer and colleagues reported a similar explanation for the possible mechanism underlying the cell origin and phenotypic detection of primary liver cancer.²⁸ They demonstrated that the same cell type, such as hepatocytes, can lead to different types of tumors, depending on the manner of cell death in the hepatic microenvironment during carcinogenesis. This finding supports the impact of different types of microenvironments on the phenotypes of primary liver cancers.

In the study from Seehawer and colleagues,²⁸ tail-vein injection or electroporation techniques were used to transfer the same cancer-promoting genes into hepatocytes in mice. In the tail-vein injection group, some cells in the microenvironment of the developing tumor underwent apoptosis, and the tumor that developed was HCC. In the electro-poration group, a necroptosis that is generally known to be associated with inflammation occurred in the microenvironment, and the hepatocytes that harbored the same oncogenic driver gave rise to ICC. Interestingly, ICC was shown to originate from differentiated hepatocytes.²⁸ Similar findings were reported in clinical studies, with apoptosis being the primary type in HCC and necroptosis being the primary type of cell death in ICC.^31,32

These findings raised the question of whether hepatocytes poised to become cancer cells are undergoing epigenetic changes due to necroptosis-associated inflammation. The authors found differences in complex histone protein and chromatin structure between the 2 cancer subtypes.²⁸ The mechanism of how and when these differences developed is unknown.

However, this type of difference may have long-term results on gene expression. For example, with the assumption that the signaling pathways developed by the induction of cell death processes affect the chromatin structure of the cells that initiate tumor development, the mystery of how the same cell type can lead to different tumors with different morphology and prognosis could be explained.

Different types of cytokine releases were noted between tail-vein injection and electroporation groups. Cells undergoing necroptosis were shown to release DAMPs that can shape the hepatic microenvironment. Specific cytokines were secreted from immune cells that are activated by DAMPs released from necroptotically dying hepatocytes. Compared with the HCC group, in the necroptosis-associated ICC group, the most differentially regulated factors brought to light with the cytokine expression profiling were Ccl4, Aimp1, Cxcl13, Ccl6, Ccl8, Pf4, and Osm. In addition, the necroptosis-associated hepatic cytokine microenvironment switched from HCC to ICC independently of the oncogenic drivers.²⁸

Cytokines released by hepatocytes undergoing apoptosis and necroptosis might act as drivers of the switch of cancer types from HCC to ICC and ICC to HCC. Thus, the release of such cytokines because of tissue injury and cell death may shape the identity and prognosis of cancer.

To further determine whether all of these cytokines were genuinely linked to necroptosis-associated cell death in electroporation-treated livers, the authors used necrostatin 1, a potent inhibitor of RIPK1 and suppressor of necroptosis. The administ-ration of necrostatin 1 inhibited necroptosis, shifted death from necroptosis to apoptosis, and reduced electroporation-induced cytokine release. Surprisingly, ICC induced by necroptosis resulting from electro-poration converted to HCC with necrostatin 1 treatment.²⁸

To summarize, liver carcinomas inevitably develop after chronic liver injuries due to inflammatory processes, such as viral hepatitis and steatohepatitis. During the inflammatory process of the liver injury, distinct types of cell death, such as apoptosis, necroptosis, or necrosis, will occur.

The liver cancer type changed from ICC to HCC when cell death in the hepatic microenvironment switched from necroptosis to apoptosis. For hepatocytes that died by necroptosis, the necroptosis-dominated microenvironment led to the development of ICC. However, for hepatocytes that died by apoptosis, the apoptosis-dominated microenvi-ronment promoted the development of HCC. The most crucial point that should not be overlooked is that the shift is independent of the oncogenic driver and occurs at the stage of tumorigenesis, not when the tumor has already developed.

The cytokine content in the necroptosis-dominant liver microenvironment is very different from that of other cell death-related microenvironments. The necroptosis-associated hepatic cytokine microenvi-ronment has been shown to switch from HCC to ICC. Thus the features of cytokines in the hepatic microenvironment during chronic injury might shape the identity and prognosis of cancer. This also implies that, during a chronic injury in the liver, changing the cytokine profile of the hepatic microenvironment could allow a shift toward a less fatal condition with improved prognosis, changing the natural course of disease progression.

Altogether, the evidence has suggested that, during chronic inflammation and injury, the hepatic microenvironment generates ingenious signals that may induce long-lasting changes in the cancer-forming cells.

Necroptosis in Tumor Growth and Tumor Metastasis
The role of necroptosis in cancer development and progression is complicated and mysterious. Necroptosis plays a pivotal role in regulating carcinogenesis, cancer subtypes, cancer immunity, cancer metastasis, and anticancer treatment.2,5,6,8 The necroptosis process is a double-edged sword in cancer. Because of the dual effects of necroptosis on cancer biology, on the one hand, necroptosis-associated inflammation promotes cancer progression and metastasis. On the other hand, necroptosis also acts as a rescue mechanism in the development of adaptive immune responses that can defend against tumor progression.2,5-9,16,22 In cases where apoptosis is inhibited, necroptosis can prevent tumor development.6

Release of necroptosis-associated DAMPs may also recruit immune and inflammatory cells that may induce tumor development by stimulating angio-genesis and cell proliferation and augmenting tumor growth and cancer metastasis.6,16 The induction of autophagy by necroptosis can further improve the invasion efficacy of metastatic cells by strengthening their fitness against necroptosis. Necroptosis-associated inflammatory cells in the tissue microenvironment may also release reactive nitrogen intermediates and reactive oxygen species (ROS) that will damage DNA and lead to genomic instability, thereby facilitating carcinogenesis.22,33

Metastasis is the primary cause of death in cancer patients. The role of necroptosis in the generation of tumor metastasis is not fully understood. Decreased expression of critical molecules in the necroptosis-related pathways has been shown in various cancers, suggesting that tumor cells may evade necroptosis to survive and lead to metastasis.

Both RIPK1 and RIPK3 modulate necroptosis and therefore may also control cancer progression.⁶ The tumor-suppressing effects of RIPK3 have been especially well documented. In animal studies, RIPK3 knockout mice were at a higher risk of developing colorectal cancer and breast cancer.^6,34,35 Reduced or loss of RIPK3 expression has been shown to have a significant prognostic value. Decreased RIPK3 expression has been shown in numerous human tumors, such as primary liver carcinoma, leukemia, melanoma, colorectal carcinoma, and breast carcinoma, indicating a worse prognosis.⁶ The downregulation of RIPK1 and RIPK3 has been shown to correlate with tumor progression and metastasis.^6,34,35 Decreased disease-free survival and overall survival were also reported in cancers with low RIPK1 and RIPK3 expression.⁶ Previous reports have indicated that metastasis and necroptosis association can exhibit duality.^6,17,36,37 Expression of necroptosis-associated RIPK1 and RIPK3 molecules has been reported in most cases with HCC and ICC, indicating the presence and activation of the necroptotic cascade in the tumor microenvironment of liver tumors.^28,32,38 However, RIPK1 and RIPK3 expression intensities were lower in HCC and ICC tumor tissues than in normal liver tissues.^39,40 Moreover, the expression of RIPK1 and RIPK3 in liver tumor cells was inversely correlated with the adverse prognostic factors of HCC and ICC, such as perineural invasion and nodal metastasis.^32,38-40 In addition, the expression of RIPK3 correlated significantly with tumor-node metastasis stages. Overall survival was also significantly increased in patients with HCC and ICC who had higher RIPK3 and RIPK1 expression levels.^32,38-40

Altogether, these reports show that necroptosis plays a preventive role in the progression and metastasis of cancer. In support of this, the necroptosis-associated effects of shikonin were shown to markedly reduce both the primary tumor and the lung metastasis of osteosarcoma.⁴¹ Shikonin, a Chinese herbal medicine, is a natural naphtho-quinone pigment purified from Lithospermum erythrorhizon. Shikonin was shown to reduce metastasis of osteosar-coma by induction of RIPK1- and RIPK3-dependent necroptosis.⁴¹ The antimetastatic characteristics of RIPK1- and RIPK3-dependent necroptosis by shikonin were also shown in other tumors, such as HCC, glioma, and breast carcinoma.^42-44 Shikonin was shown to kill glioma cells through necroptosis mediated by RIPK1.⁴³

In addition to the RIPK pathway, oxidative stress also participated in the activation of shikonin-induced necroptosis. Reactive oxygen species is recognized as an essential mediator in the devel-opment of necroptosis, and RIPK3 has been shown to regulate ROS under necroptotic conditions, preventing metastases by mediating oxidative stress, which is critical in killing metastatic cells.^42-44 Accordingly, necroptosis can be used as a critical pathway that can prevent tumor metastases.

Necroptosis may need to be inhibited by tumor cells to provide successful tumor progression and metastasis. In support of this, tumor cells were shown to develop resistance to necroptosis to maintain their survival.45 In this context, studies are being conducted on new molecules that can induce necroptosis in tumor cells to overcome such resistance against necroptosis.

In contrast to these observations, however, upregulated RIPK1 and RIPK3 have been shown to play an oncogenic role in tumorigenesis.^8,35,36 Under certain circumstances, necroptosis might promote metastasis.^8,17,35,36 Various tumors, such as glioblas-toma, head and neck squamous cell carcinoma, breast carcinoma, lung cancer, and pancreas cancer, have been reported to show poor prognosis with upregulation of RIPK1 and RIPK3 in the tumor.^{6,17,35,36,46,47}

Cancer cells may cause necroptosis in endothelial cells via the activation of death receptor 6 (DR6), causing extravasation of tumor cells from the vessels.¹⁷ As a result, cancer metastasis may occur. When DR6 binds to its ligand amyloid precursor protein, endothelial cell death and tumor cell extravasation may occur. Strilic and colleagues reported that endothelial cells undergoing necroptotic death provide a tunnel where tumor cells can pass and start to extravasate through vessel walls to tissues.¹⁷ The DAMP molecules released by necroptotic cells may promote extravasation and metastasis of tumor cells by affecting tumor cells and adjacent endothelial cells.¹⁷

To summarize, the role of necroptosis on carcinogenesis, tumor growth, and metastasis is complicated, and it remains unclear whether necroptosis facilitates or suppresses tumor growth and metastasis. The role of necroptosis may vary according to the different tumor microenvironments or biological characteristics of each tumor type. More reliable data are needed on the pathophysiological and molecular functions of the necroptotic pathway in order to unravel the mysteries of necroptosis on cancer development and progression.

Necrosis and Primary Liver Cancer
Necrosis is another type of cell death in damaged livers with the development of carcinoma. Cancer cells are known to be resistant to apoptosis as a result of p53 mutation.^4,6,45 Therefore, necrosis is the primary cell death pathway in cancer therapy.^3-5,45 Necrosis is associated with high levels of angiogenesis, tumor-associated macrophages, and increased inflammation in the tumor microenvironment.^48-50

Tumor necrosis may develop spontaneously within the tumor or may occur due to anticancer treatment.^51-54 Although the causes of spontaneous necrosis are still unclear, some possibilities leading to spontaneous necrosis in liver cancers include the use of herbal medicine, blood supply cessation, vascular injury secondary to angiography, rapid tumor growth, immunological reactions, cessation of drinking, and androgen therapy.^51-57 In the resection specimens of livers after spontaneous necrosis, inflammatory cells, including acquired and naive immune cells, were detected in the necrotic tissue. It has been suggested that activation of host immune cells could be the most critical factor for spontaneous regression of the tumor.^51-57

Only little is known on whether the presence and the degree of spontaneous necrosis in liver cancer are potentially related to other critical histological features of tumors, such as vascular invasion. The presence of tumor necrosis was reported in 50% of patients with HCC, and tumor necrosis in these cases was significantly correlated with tumor size and vascular invasion.^50,52-56

Tumor necrosis in patients diagnosed with HCC and ICC is associated with overall and recurrence-free survival, and it is more common in tumors with progressively worse overall characteristics.^54,58 In HCC, tumor cells with more than 50% necrosis were able to upstage smaller and more favorable tumors. Regarding survival, stage T1 HCC tumors with >50% necrosis had survival equivalent to a stage T2 HCC tumor, and T2 HCC tumors with >50% necrosis became indistinguishable from stage T3 HCC tumors.⁵⁸

Surgical resection of liver tumors is generally not feasible, as most patients present with advanced tumor stage. It has been reported that only 10% to 20% of patients can have surgical resection of the liver tumor.^54,59 Generally, neoadjuvant therapy is not recommended routinely before the resection of HCC. Instead, local therapies like radiofrequency ablation (RFA), transarterial chemoembolization (TACE), and transarterial radioembolization modalities are frequently preferred to bring the tumor to the resectable stage before surgery.^54,59 All of these modalities are known to generate significant diffuse necrosis in the tumor.

Thus, treatment-related necrosis can also cause cytotoxicity in tumor cells, resulting in accelerated necrosis in the tumor. The content of cells undergoing necrosis triggers various cytokine releases that have roles in cancer progression via inflammatory and noninflammatory pathways.

However, one critical question should be answered: Could tumor necrosis in liver tumors following these therapy modalities negatively influence patient survival? Rapid intrahepatic neoplastic progression after local treatment was reported in 4.5% of patients.⁶⁰ Local tumor progression without extrahepatic metastasis was observed in 5 cases, 1 after TACE and 4 after RFA. Seki and colleagues suggested that, although most tumor cells underwent necrosis after TACE, viable tumor cells may be left in the liver.⁶¹ Thus, this partial necrosis of the tumor induced by TACE weakens the adhesive potential of the tumor cells and causes the dissemination of these tumor cells.⁶¹ Furthermore, Adachi and colleagues suggested that, because the remaining tumor cells are less firmly attached and more likely to leak into the bloodstream, manipulating a partially necrotic tumor may lead to recurrences and progression.⁶²

To summarize, these results have shown that tumor necrosis in liver cancers generated spontaneously or by anticancer therapy is a critical parameter in determining the destiny of the tumor. Tumor necrosis that develops spontaneously shows aggressiveness of the tumor and poor prognosis. Although necrosis developing after treatment is a desirable condition for the destruction of the tumor cells and for better prognosis, it should be noted that necrosis that develops rapidly after treatment may induce tumor progression and recurrence due to the destabilization of the tumor cell stroma and cell-cell contact.

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