Cancer caused by too much apoptosis-An intriguing contradiction?
Authors: Philipp J. Jost, Thomas Kaufmann
Journal: Hepatology (2009)

Apoptosis (a crucial physiological form of programmed cell death) of hepatocytes is a critical prerequisite to preserve liver homeostasis and protect against malignant transformation and carcinogenesis. In a report by Weber et al. in this issue of HEPATOLOGY,1 the authors identify the prosurvival B cell lymphoma-2 (Bcl-2) family member myeloid cell leukemia-1 (Mcl-1) as a critical player in hepatocyte apoptosis. Interestingly, the authors provide data that the increased spontaneous apoptosis observed in hepatocytes lacking Mcl-1 translates into development of malignant hepatocellular carcinoma (HCC)-like lesions in mice starting from 8 months of age. Using a mouse model harboring the loxP-targeted allele of Mcl-1 in addition to Cre recombinase expressed under the albumin promoter, the study provides convincing, genetically precise experimentation and an intriguing finding: increased apoptosis in hepatocytes goes hand in hand with carcinogenesis illustrating an intriguing connection between apoptosis and cancer research. Bcl-2, B cell lymphoma-2; BH3, Bcl-2 homology domain 3; Bid, BH3-interacting domain death agonist; HCC, hepatocellular carcinoma; Mcl-1, myeloid cell leukemia-1. The critical role of the prosurvival Bcl-2 family members Mcl-1 and Bcl-x(L) in guarding apoptosis in hepatocytes has previously been described by the same group and others.2-4 Mice lacking either Mcl-1 or Bcl-x(L) (both constitutively expressed in the liver) specifically in their hepatocytes present with strongly increased spontaneous hepatocyte apoptosis and liver fibrosis. Furthermore, Mcl-1–deficient hepatocytes are more susceptible to Fas-mediated liver damage.2 Mcl-1 and Bcl-x(L) cooperatively regulate hepatocyte integrity to a point where liver-specific deletion of both proteins leads to rapid postnatal death of experimental animals due to hepatic failure.3 The findings on the apoptotic function of Mcl-1 and Bcl-x(L) are interesting, but somewhat expected, especially in the light of their known antiapoptotic function and their expression pattern in the liver. Nonetheless, the story of Bcl-2 family members in liver homeostasis becomes exciting with the publication of the recent report by Weber and co-workers defining the function of Mcl-1 in carcinogenesis.1 The authors show that aged Mcl-1fl/fl–AlbCre mice spontaneously develop HCC-like lesions, which occur with an incidence of greater than 50% at the age of 8 and 12 months. They further present evidence that the chronic liver damage and HCC formation observed in those animals occurs in the background of fibrosis but in the absence of apparent inflammatory responses, indicating that liver carcinogenesis is promoted by enhanced and "clean" apoptosis. In their seminal review in 2000, Hanahan and Weinberg described the hypothetical connection between apop- tosis and carcinogenesis. They stated that increased apoptosis should protect from malignant transformation rather than increase malignant transformation.5 The intracellular stresses that are induced by malignant transformation are highly apoptogenic (including lack of oxygen and nutrients, violation of cell cycle checkpoints, and others). Transformation therefore forces expression of apoptogenic factors including proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins from the Bcl-2 family (e.g., Bid [BH3-interacting domain death agonist], Bim, Bad [Bcl-2–associated death promoter], Noxa, Puma [p53 up-regulated modulator of apoptosis]). Thus, induction of apoptosis constitutes a mechanism by which a cell protects itself against transformation and, in turn, blocking the apoptotic machinery represents a potential mechanism of a cell to survive neoplastic transformation. The description of spontaneous tumor formation in cells or tissues with increased apoptotic susceptibility is therefore counterintuitive, adding another level of complexity to the interplay between apoptosis and carcinogenesis. If the concept of elevated malignant transformation in a proapoptotic environment holds true also for tissues other than the liver, the impact could be enormous. Until now, the main focus of antineoplastic drug development using proapoptotic compounds strongly depended on the notion that more apoptosis means less malignant growth. With those new insights, however, we might have to rethink the applicability of proapoptotic molecules including BH3-mimetic compounds as anticancer agents. Although certainly more data is needed to support these findings, their mere possibility is challenging for researchers and drug companies alike. What connection exists between increased apoptosis and malignant transformation in hepatocytes lacking Mcl-1? The report by Weber and coworkers offers a potential explanation: the hyper-apoptotic environment in hepatocytes lacking Mcl-1 in aged mice correlates with elevated proliferation within the liver, as shown by increased bromodeoxyuridine and Ki67 staining (see figure 3, C and D, in Weber et al.1). In addition, the chronic liver injury caused pericellular fibrosis, demonstrating a link between apoptosis induction and fibrinogenesis. The increase in proliferation presented is not dramatic, but taken into account that unchallenged livers are shown, might constitute a critical component toward carcinogenesis (Fig. 1B). The authors deduct from this finding that increased proliferation is a consequence of increased apoptosis. However, the direct link between these two separate cellular fates is not entirely clear. It is known that some primarily apoptotic proteins including caspases or FADD (Fas-associated protein with death domain) exert nonapoptotic functions like cellular activation, proliferation, and differentiation (reviewed in Lamkanfi et al.6 and Strasser et al.7). However, the findings are somewhat contradictory, especially in the liver. The detailed understanding of nonapoptotic functions of caspases (or FADD) including the precise mechanisms, upstream activators, and downstream targets will tremendously help cell death researchers and potentially foster future antineoplastic drug development (reviewed in Strasser et al.7). Diagram depicts how Mcl-1 regulates apoptosis in hepatocytes and how its absence might contribute to malignant transformation. (A) Apoptotic stress stimuli, e.g., induced by metabolic or genotoxic stress, lead to the activation of BH3-only proteins, which trigger mitochondrial outer membrane permeabilization (MOMP) in a Bak-dependent or Bax-dependent manner, leading to downstream caspase activation and apoptotic death. Mcl-1 and its relative Bcl-x(L) inhibit MOMP and apoptosis by neutralizing Bak by direct interaction and by acting as a sink for activated BH3-only proteins. (B) Mice lacking Mcl-1 in hepatocytes, on the other hand, present with increased spontaneous hepatocellular apoptosis and chronic liver damage, presumably due to a low but constant pool of activated BH3-only proteins (e.g., Bid or Bim). Increased apoptosis correlates with increased proliferation as well as fibrinogenesis by some ill-defined mechanism and may result in compensatory events (such as up-regulation of Survivin?) favoring the selection and outgrowth of malignantly transformed cells, leading to HCC. Another possible explanation of how increased apoptosis translates into carcinogenesis might be found in the up-regulation of Survivin, an inhibitor of apoptosis (IAP) family member that the authors found to be up-regulated in hepatocytes lacking Mcl-1 (see figure 2, C and D, in Weber et al.1). Survivin is involved in cell cycle progression, proliferation, assembly of the mitotic spindle, but also apoptosis (reviewed in Altieri8). Survivin is highly expressed in a multitude of cancers, independently of their mitotic index, indicating that it is an interesting candidate to drive cell division and cell cycle entry. Its expression, however, might be a consequence (or unrelated effect) of increased proliferation rather than its cause. The study by Weber et al. would benefit from functional data on the role of Survivin; however, genetically accurate testing involves breeding Survivin-deficient mice into the Mcl-1fl/fl–AlbCre background. Defining the key players that drive proliferation in Mcl-1–deficient hepatocytes constitutes an intriguing and difficult task ahead. Solving the key components of potential caspase-mediated proliferation will offer a variety of new insights into liver homeostasis and probably other tissues as well. What is the apoptotic stimulus that kills Mcl-1–deficient hepatocytes in unchallenged mice? The mitochondrial permeabilization by active Bak or Bcl-2–associated X protein (Bax) within hepatocytes is critical for intrinsic cell death progression. Although it is not clear which prosurvival Bcl-2 family members exactly neutralize Bax, its homologue Bak is subject to negative regulation specifically by Mcl-1 and Bcl-x(L) but not other prosurvival family members.9 Mcl-1 guards Bak and shields the outer mitochondrial membrane from apoptotic stimuli transmitted by BH3-only proteins such as Bim, Puma, and Noxa (Fig. 1A). The report by Weber et al. presents data on apoptosis (together with data from Vick et al.2) but does not provide evidence on the upstream stimuli that induce cell death. One might speculate that metabolic stress within the liver constitutes a "steady-state" stimulus sufficient to activate a relatively low but constant number of BH3-only proteins. Precise knowledge of which BH3-only proteins are activated in hepatocytes might help to identify the upstream stimulus and would conclude an already exciting picture of how apoptosis proceeds in Mcl-1–deficient hepatocytes (Fig. 1A). The mechanistic insight provided by the authors lacks data on a likely posttranslational regulation of BH3-only proteins. Firstly, the BH3-only protein Bim might be involved in apoptosis initiation in hepatocytes lacking Mcl-1 because it has been shown to mediate an important (albeit only partial) aspect of TRAIL (TNF-related apoptosis-inducing ligand) and tumor necrosis factor-α induced apoptotic response in the liver.10, 11 Secondly, for many BH3-only proteins, including Bim and Bid, posttranslational regulation is equally if not more important compared to transcriptional regulation (reviewed in Bouillet and O'Reilly12 and Puthalakath and Strasser13). Lastly, Hikita et al. recently reported the near normal appearance of Bid−/−Mcl-1fl/fl–AlbCre livers in young adult mice, identifying Bid as an important apoptotic mediator in hepatocytes lacking Mcl-1.3 It will be interesting to see whether those same mice are less prone to HCC development than their Bid-proficient littermates as they age. What is the cause of malignant transformation? Is genomic instability a consequence of elevated proliferation and sufficient to drive carcinogenesis? The authors show an increase in genomic instability in hepatocytes of HCC-like lesions from Mcl-1fl/fl–AlbCre mice. This finding supports the concept that the tumor nodules indeed possess a malignant phenotype and that a high degree of genomic instability is present. However, the question yet again arises whether this is the cause or the consequence of transformation. Another interesting open question is why Bcl-x(L)fl/fl–AlbCre mice, which share a very similar phenotype as Mcl-1fl/fl–AlbCre mice at young age, including increased hepatocellular apoptosis and fibrinogenesis, do not seem to develop malignant HCC-like lesions.4 In conclusion, the study by Weber et al. presents clear-cut genetic data on the function of Mcl-1 in aged mice. It provides evidence that increased levels of apoptosis translate into elevated proliferation and malignant transformation of hepatocytes, which is pronounced as experimental animals age. The understanding of apoptosis initiation in the liver profits from the presented data, and it provides the basis for identification of the exact molecular events linking apoptosis and carcinogenesis in this model, of which not only hepatologists but also cell death researchers in general will greatly benefit.