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Successful stories of drug repurposing for cancer therapy in hepatocellular carcinoma
Hepatocellular carcinoma
Hepatocellular carcinoma (HCC) is a major health problem with devastating consequences associated with treatment failure. It has an estimated global incidence of more than 850,000 new cases annually [ ]. HCC is currently the second leading cause of cancer-related death worldwide and accounts for 90% of cases with primary liver cancer [ ]. Several risk factors contribute to HCC development such as liver cirrhosis, viral hepatitis including hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, fatty liver, and alcohol abuse. Smoking and the fungal carcinogen, aflatoxin B1, are also well-known contributors to HCC [ ]. Recent advances in HBV vaccination and antiviral therapeutics have remarkably contributed to a decrease in incidence. However, nonalcoholic fatty liver disease (NAFLD) and its progressive form, nonalcoholic steatohepatitis (NASH), at its current pace of growing prevalence approaching epidemic proportions is projecting as the most common underlying etiology of HCC, presented in almost 60% of cases, which made NAFLD-associated HCC an emerging indication for liver transplantation [ ]. Additionally, HCC has a notable gender predilection where incidence in men is threefold that in women [ , ].
Molecular pathogenesis of HCC
Over the past decade, substantial progress has been achieved in understanding how HCC develops and progresses in an attempt to improve treatment options [ ]. HCC is a very heterogeneous disease in terms of both phenotype and genotype. This heterogeneity could be attributed, in part, to the divergent nature of the contributing factors, the complexity of the liver microenvironment, and the stage at which HCC turns to be clinically evident/detectable.
Malignant transformations in liver cells are driven by several factors such as chronic injury or inflammation due to oxidative stress that may lead to genetic and epigenetic modification. These modifications consequently lead to disrupted cellular signaling pathways leading to an overexpression in several growth factors and their receptors. This inevitably results in cell resistance to apoptotic signals, stimulation of angiogenesis, and uncontrollable proliferation besides the acquisition of a metastatic phenotype [ , ].
Since HCC almost exclusively develops in patients with chronic liver diseases, injury of liver cells can promote the progression to HCC over a long period of time [ ] driven by a number of cytokines and inflammatory mediators along with aberrant activity of several signaling pathways, as shown in Fig. 8.1 (will be discussed later).
Therapeutic management of HCC
HCC mostly exhibits resistance to conventional chemotherapy. Besides, patients with HCC are usually intolerant to treatment due to an underlying hepatic dysfunction. However, almost half of HCC patients still receive chemotherapy at some point during the course of the disease [ , ]. Chemotherapeutic intervention mainly relies on the multitargeted tyrosine kinase, sorafenib, that acts by effectively blocking the Ras/Raf/MAPK pathway impeding cancer cell ability to circumvent apoptotic signals and induce angiogenesis, proliferation, and invasion. Sorafenib was found to extend the median survival in advanced HCC patients for up to 3 months with manageable adverse effects; however, it lacks predictive biomarkers to reflect on responsiveness [ ]. Sorafenib remained the only approved systemic treatment for HCC between 2007 and 2016. Yet, promising outcomes were reported in randomized phase III trials using other multiple kinase inhibitors such as lenvatinib [ ], regorafenib [ ], cabozantinib [ ], and ramucirumab [ , ], where regorafenib has received FDA approval in the second-line setting. Moreover, nivolumab, a monoclonal immunotherapy-based antibody targeting the immune checkpoint programmed cell death protein 1, showed also positive response rates and mean overall survival durations in a phase I–II trials performed on patients who were formerly treated with sorafenib [ ] for which it has been successfully granted an accelerated FDA approval. On the other hand, several other kinase inhibitors such as sunitinib and erlotinib failed to show comparable improved survival rates especially in unrespectable HCC patients [ ].
Sorafenib efficacy over other proposed interventions is most likely attributed to its ability to target several molecules and pathways in tumor cells as well as the microenvironment. Also, the heterogeneous nature of HCC may limit the efficacy of other targeted therapeutic approaches possessing higher selectivity [ ].
HCC is undoubtedly a resistant type of cancer making treatment more challenging. Despite the fact that systemic therapy enhanced survival rates in HCC patients, therapeutic outcomes are still incremental and inadequate, especially if compared to other types of cancer. Sorafenib was the first FDA-approved drug for treatment of patients with advanced HCC for its ability to increase the median overall survival. However, with the use of the newly developed and approved multiple kinase inhibitors, the median overall survival still remains almost 1 year. Moreover, de novo resistance developing to sorafenib has been recently heavily reported impeding its beneficial clinical applications [ ]. Resistance to sorafenib involves a cross talk between several pathways such as Janus kinase/signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), and hypoxia-inducible pathways beside others [ ]. Providing new treatment options for HCC, therefore, still remains an unmet medical need. Accordingly, further insights into the molecular targets affiliated with the pathophysiology of HCC will be dissected in the following section.
HCC drivers and affiliated molecular targets
To date, we still have very few identified “druggable” drivers for HCC. However, most of the preclinical and clinical attempts to identify pharmacological interventions, whether new or repositioned ones, focused on signaling pathways implicated in disease progression. Hence, investigating effect on oncogenic drivers for the identification of new molecular targeted therapies in HCC needs to be revisited. In order to provide pharmacological interventions for HCC, the main drivers and pathways involved have to be identified first. As it is the case with solid tumors, a simultaneous alteration in at least three signaling pathways and five to eight driver genes should take place for HCC to develop [ ].
The main drivers and pathways contributing to HCC development and progression will be discussed in the following section in more details. These include, but not limited to, Ras/Raf/MAPK, PI3K/AKT/mTOR, Wnt/β-catenin, hedgehog (Hh), and IL-6/STAT3 pathways. Moreover, the role of the following key players will be explored: tumor necrosis factor-alpha (TNF-α), nuclear factor kappa-B (NF-κB), c-Jun N-terminal kinase (JNK), gut microbiome, and toll-like receptors and also adaptive immunity. All these molecular targets and signaling pathways represent potential candidates for targeting HCC, as shown in Fig. 8.2 .
Ras/Raf/MAPK pathway
Among the most extensively investigated pathways implicated in HCC development and progression is the Ras/Raf/MAPK pathway. Cell surface receptor tyrosine kinases such as insulin-like growth factor (IGF) receptor, endothelial and vascular epidermal growth factor receptor (EGFR), c-Met, and platelet-derived growth factor receptor send signals that are transmitted to the nucleus via this pathway to control cell survival, growth, and differentiation. Upregulation of the Ras/Raf/MAPK pathway in liver cells induces cell growth augmenting antiapoptotic signals leading ultimately to HCC [ ]. Aberrant upstream IGF and EGFR signaling, suppression of Raf kinase inhibitor protein, and HBV- and HCV-related proteins induction are among the mechanisms by which the Ras/Raf/MAPK pathway can be activated toward HCC development [ ].
PI3K/AKT/mTOR pathway
Mounting evidence over the past years suggested the aberrant upregulation of PI3K/AKT/mTOR pathway in HCC where mTOR phosphorylation and a subsequent upregulation of its downstream effector, p70S6k, were reported in almost 50% of HCC patients [ ]. Likewise, almost 40% of patients encountered an activated mTOR [ ]. Aberrant PI3K/Akt/mTOR pathway was also correlated with poor prognosis, especially in advanced-stage HCC [ ]. The underlying mechanism by which PI3K/AKT/mTOR pathway is activated in HCC is not yet fully deciphered, yet overly expressed upstream IGF, c-Met, or EGFR is likely [ ]. Hepatitis viral infections are also capable of activating PI3K/AKT/mTOR pathway in liver [ ], where HCV infection was found to increase neuroblastoma (N)-Ras expression in HCC, which turns on the PI3K/AKT/mTOR pathway [ ]. Loss of PTEN , a tumor suppressor gene, by mutations was also evident in HCC patients [ ], contributing to AKT activation. Apart from PTEN , mutations in PI3K catalytic subunits were also reported [ ]. Based on the previously presented sound evidence from previous studies, different agents that target the main key players of this pathway such as PI3K, AKT, or mTOR are being tested on HCC animal models and others currently enrolled in clinical trials [ , ].
Wnt/β-catenin pathway
The Wnt pathway comprises a canonical or β-catenin–dependent pathway as well as a noncanonical or β-catenin–independent one. The classic or canonical pathway consists of the Wnt protein and Wnt protein ligand, also known as frizzled protein, besides other proteins such as β-catenin and glycogen synthase kinase 3 [ , ]. Aberrant activation of Wnt/β-catenin pathway was found to support tumor growth. When the upstream Wnt protein complexes with its ligand, β-catenin builds up in cells and gets transferred to the nucleus where it dimerizes with LEF/TCF, a downstream transcription factor that regulates the transcription of other genes such as cyclin D [ ]. Abnormal upregulation of Wnt/β-catenin pathway was linked to HCC development and cancer stem cell maintenance. Aberrant β-catenin was detected in almost 90% of liver cancers [ ].
Hedgehog pathway
Hh signaling pathway is among the most important pathways implicated in HCC. It consists of Hh ligand, Ptch and Smo (two transmembrane receptors), and Glib nuclear transcription factor along with downstream genes. Once activated, Hh ligands bind to Ptch receptors blocking Ptch inhibitory effect on Smo. Smo then moves to the cytoplasm activating Gli, to induce specific genes upregulation, thereby controlling growth and division. The Hh pathway is rarely activated in normal liver cells; however, in HCC, it is abnormally active [ , ]. In HCC, the inhibition of the Gli2 gene can also cause Bcl-2 and c-Myc downregulation, while increasing the expression of p27, turning off the cell cycle, and thus impeding tumor growth [ ].
NF-κB and JNK
TNF-α is a pivotal protumorigenic cytokine as it is capable of stimulating both NF-κB and JNK signaling pathways [ , ]. It is currently widely believed that inflammation is the fuel that feeds the genetic aberration sparks that inaugurate the tumorigenic process. Moreover, the wide spectrum of chronic injury etiologies in the liver can sufficiently act as both initiators and promoters of HCC. In this last decade, several inflammatory mediators were identified and their roles were extensively elucidated in the pathogenesis of chronic liver disease [ ]. Most of these inflammatory mediators act as activators or targets for NF-κB [ , ]. NF-κB is considered a key transcriptional factor for almost all chronic liver diseases such as viral hepatitis, alcoholic liver disease, and NASH [ ]. It also finely tunes crucial functions in liver cells such as Kupffer cells (KCs; hepatic resident macrophages) and hepatic stellate cells (HSCs). Inhibition of NF-κB–related signaling cascades, however, may lead to liver fibrosis and tumorigenesis and that is why NF-κB is considered very essential in maintaining homeostasis and wound-healing processes in the liver [ ]. Accordingly, NF-κB is thought of as a two-edged sword since inhibition may be sometimes beneficial; however, it may also influence other essential processes pertaining to liver homeostasis.
Regarding JNK, two isoforms are expressed in liver cells, namely, JNK1 and JNK2, where the former being linked to carcinogenesis. JNK1 protumorigenic potential is defined by its ability to induce the proliferative potential of HCC [ ]. It also regulates the proliferation of HCC cells via downregulation of p21 and upregulation.of c-Myc [ ]. Moreover, apoptosis induced by caspase-8 was found to activate JNK and hence promote proliferation of liver cells [ ]. These findings propose that hepatocyte apoptosis is what triggers JNK activation. Although, the role of JNK in HCC is controversial, JNK1 and 2 knockout attenuated HCC in an animal model of DEN, exhibited p21 upregulation, and decreased c-Myc [ ]. Overall, TNFα, NF-κB, and JNK pathways can either have prosurvival or proapoptotic potentials augmenting HCC proliferation and growth. Thus, treatment approaches involving NF-κB and JNK inhibition should act moderately to avoid a complete shutdown in hepatocytes leading to liver injury and subsequent HCC.
IL-6/STAT3 pathway
IL-6, as a mediator of STAT3 activation, is an essential driver of liver cell proliferation, which may consequently lead to HCC [ ]. Moreover, overactive STAT3 coinciding with high levels of IL-6 was found in patients with HCC [ ]. Likewise, STAT3 upregulation had promoted DEN-induced HCC experimentally [ ]. IL-6 autocrine production is essential for malignant transformations in HCC, which once develops, IL-6 paracrine production from KCs start initiating growth and proliferation of HCC cells [ ]. Activation of STAT3 may also take place via IL-22, produced by Th17 cells, that is overly expressed in HCC patients. Similar to IL-6, IL-22 also plays a role in promoting DEN-induced HCC in mice via STAT3 pathway [ ]. Besides IL-6 and IL-22, IL-17 can also activate STAT3. This action, however, is IL-6-dependent [ ] Collectively, these findings propose a role for IL-6/STAT3 pathway in promoting hepatic carcinogenesis suggesting it as a potential therapeutic target for treatment of HCC.
Innate and adaptive immunity
Overwhelming evidence from the past few decades have underlined the role of dysbiosis in the development of chronic liver disease and HCC. Moreover, the role of activating the innate immune system–related receptors in general and toll-like receptors (TLRs) in particular, in HCC development, and progression was also explored [ ]. Due to its anatomical location, the liver is considered the first organ exposed to gut-derived microbial products translocated through the portal vein that in turn leads to TLR activation in the liver [ ]. TLR-4 is expressed in different liver cells such as KCs, HSCs, and hepatocytes. It was previously reported that activation of TLR-4 on liver cells by bacterial lipopolysaccharides, a gram-negative cell wall component, leads to subsequent fibrotic and carcinogenic events [ ]. The immune surveillance hypothesis suggests that the immune system may protect against nascent tumors by destroying malignant cells early on, before they develop into detectable tumors. The T-cell antitumor role is facilitated through immune surveillance by CD4+ and CD8+ T cells. Interestingly, an animal model of NASH-associated HCC was found to cause a depletion of CD4+ T cells impairing immune surveillance [ ]. Other studies, however, reported a protumorigenic potential of CD8+ T, natural killer T cells, and T-helper (Th) 17 cells in an animal model of choline-deficient high fat diet [ ]. Yet still debatable, these findings suggest a role for adaptive immunity in HCC development and progression. Thus, targeting it in the appropriate context may offer a chemopreventive strategy for HCC patients.
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