The MAPK intracellular signaling pathway, which is mainly involved in cell growth and survival, and regulates cell differentiation, is upregulated in cancer cells. Therefore, this pathway has been extensively studied as a therapeutic target. The MAPK pathway is a common downstream pathway for the EGFR, PDGFR and VEGFR, and is universally used for signal transduction downstream of cytokine receptors, integrin complexes, and G-protein receptors to Ras. The MAPK pathway also plays an important role in HCC, in that its activation is reportedly involved in HCC growth and survival. The downstream ERK is activated by two upstream protein kinases, which are coupled to growth factor receptors by Ras proteins. Ras, which is activated by ligand binding, activates Raf serine/threonine kinases and MEK (MAP kinase/ERK kinase), while MEK phosphorylates and activates ERK, which phosphorylates proteins involved in cell growth, apoptosis resistance, extracellular matrix production and angiogenesis[13-15].

Raf and Ras inhibitors: Raf and Ras are proto-oncogenes. In particular, K-Ras mutations are commonly observed in many cancers, including pancreatic and colorectal cancers. One study reported that 30% of HCCs have Ras mutations[16]. To our knowledge, no agents targeting Ras are planned to enter clinical trials at the present. However, because the binding of Ras protein to the cell membrane and its functional activation require farnesylation, several farnesyltransferase inhibitors are being tested for Ras-related tumors. In addition, vaccine therapy for mutant Ras proteins is currently being tested for solid cancers, including HCC.

The Raf family consists of three isoforms, A-Raf, B-Raf and C-Raf/Raf-1. Genetic abnormalities, such as point mutations and gene rearrangements, have been reported in various cancers[17]; however, in HCC, ras/raf mutations are rare, and no k-ras or b-raf mutations have been detected[18]. On the other hand, wild-type Raf-1 was reported to be hyperactivated in many cancers, including HCC[19-21]. Sorafenib inhibits Raf, and has multiple characteristics in that it exhibits strong inhibitory activity against Raf-1 (C-Raf) kinase, B-Raf (wild-type B-Raf and mutant V600E B-Raf) serine/threonine kinase, the pro-angiogenic receptor tyrosine kinases VEGFR, PDGFR and FGFR1, and tyrosine kinases, such as c-kit, Flt-3 and RET, which are involved in tumor progression and overall prognosis[22].

MEK: The MEK family consists of MEK1 and MEK2 proteins, which specifically phosphorylate tyrosine and threonine residues, and phosphorylates downstream Erk1 and Erk2[23].

In an immunohistochemical study, MEK1/2 overexpression, ERK1/2 overexpression, and ERK1/2 phosphorylation were observed in 100% (46/46), 91% (42/46), and 69% (32/46) of HCCs, respectively. In addition, the in vitro treatment of HepG2 and Hep3B cells with MEK1/2 inhibitors inhibited cell growth and upregulated apoptosis[24].

The MEK inhibitors CI-1040, PD0325901, AZD6244, and RDEA119/BAY869766 have been tested in several cancers, including solid tumors such as HCC. Recently, the results of PhaseIof AS703026 and E6201 studies against solid tumors were reported in ASCO2010. A phase II study of AZD6244 (selumetinib, ARRY-142866) and a phaseI/II study of RDEA119/BAY869766 in combination with sorafenib are being conducted.

The PI3K/Akt/mTORpathway also plays an important role in cell growth, survival regulation, metabolism, and anti-apoptosis. The membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) is phosphorylated by PI3K into phosphatidylinositol 3,4,5-triphosphate (PIP₃), which binds to and activates the serine/threonine kinase Akt. The tumor suppressor gene product phosphatase and tensin homolog (PTEN) deleted on chromosome is antagonistic to PI3K activity. PTEN is a lipid phosphatase that dephosphorylates inositol phosphates, such as PIP₃. The inactivation of PTEN through gene deletion increases PIP₃ levels, and activates Akt, which inhibits apoptosis, leading to the development of tumors. The serine/threonine kinase mTOR is an important mediator in the PI3K/Akt pathway, which binds intracellularly to a protein called raptor or rictor, and exists as two different complexes, complex 1 and 2 (mTORC1 and mTORC2). mTORC2 (mTOR-rictor) activates Akt, while mTORC1 (mTOR-raptor) is activated downstream of Akt; thus, both molecules regulate protein synthesis (Figures 4 and 5)[25].

Open in New Tab   Full Size Figure   Download Figure

Figure 4 Phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling pathway in cell proliferation in solid cancer. PI3K: Phosphoinositide 3-kinase; mTOR: Mammalian target of rapamycin; PTEN: Phosphatase and tensin homolog; TSC: Tuberous sclerosis; FKBP12: FK506-binding protein 12.

Open in New Tab   Full Size Figure   Download Figure

Figure 5 Mammalian target of rapamycin/hypoxia-inducible factor-1/vascular endothelial growth factor signaling pathway in angiogenesis in solid cancer. HIF: Hypoxia-inducible factor; VEGF: Vascular endothelial growth factor; mTOR: Mammalian target of rapamycin; FKBP12: FK506-binding protein 12.

Inhibiting mTOR with molecules, such as RAD001, generates additive effects that accompany upstream and downstream target inhibition. Alternatively, upstream receptor inhibition is compensated for by inhibiting the downstream pathway, even if some resistance develops against receptor inhibition regardless of initial or acquired resistance. Therefore, RAD001 is a potential targeted agent for HCC.

Besides the finding that mTOR plays a key role in cell biology, it was also demonstrated that mTOR and S6K are overexpressed in 15%-41% of HCCs. mTOR inhibitors also have antitumor effects in various HCC cell lines and animal models[26-29]. Activation of mTOR is correlated with the development of HCC and recurrence after the excision of early HCC. Regulating this specific intracellular pathway (Ras-Raf pathway) with RAD001 is potentially more effective in suppressing sorafenib-resistant tumors.

A study of 528 HCC samples showed that the expression of pAkt, PTEN, p27 and S6 ribosomal protein (pS6) was a poor prognostic factor for survival[30]. A tissue microarray analysis of HCC samples revealed that the loss of PTEN and overexpression of pAkt and p-mTOR were correlated with tumor grade, intrahepatic metastasis, vascular invasion, TNM stage, Ki-67 labeling index, and matrix metalloproteinase (MMP)-2 and -9 upregulation. Meanwhile, PTEN mRNA expression in the cancerous tissue was downregulated compared with that in the non-cancerous tissue. The levels of PTEN, MMP-2, and MMP-9 mRNA expression were correlated with tumor stage and metastasis, and the levels of PTEN and MMP-9 mRNA expression were inversely correlated[31]. In an extensive analysis of 314 HCC samples in terms of mutation analysis, DNA copy number changes, mRNA levels and immunostaining, Villanueva et al[32] found that activation of the IGF pathway, upregulation of EGF, dysregulation of PTEN, and aberrant mTOR signaling were present in half of the samples, and that inhibiting mTOR activity with everolimus was effective in improved survival and suppression of recurrence.

The PI3K inhibitor RG7321 and the Akt inhibitor perifosine target the PI3K/Akt/mTOR pathway and are in early stages of clinical development, while the mTOR inhibitors everolimus (RAD001), sirolimus (Rapamune), and temsirolimus (CCI-779) are at more advanced stages of development. Everolimus is used to treat sorafenib-intolerant patients, or for patients showing disease progression after sorafenib administration. A phase III study to compare everolimus and a placebo (EVOLVE-1: Advanced Hepatocellular Carcinoma after Disease Progression or Intolerance to Sorafenib EverOlimus for Liver cancer Evaluation) and a phaseI/randomized phase II study (sorafenib + everolimus vs sorafenib alone) to test the efficacy and tolerance of sorafenib in combination with everolimus are underway. Since mTOR inhibitors exhibit cytostatic and antiangiogenic effects, they are expected to be effective in combination with other angiogenesis inhibitors, such as bevacizumab, and may be appropriate for administration after transarterial chemoembolization (TACE). Furthermore, since the mTOR pathway is stimulated by factors such as EGFR, PDGFR, and TGFα, and is closely related to other signaling pathways, including the Ras/Raf/MEK/ERK pathway, they are likely to show promising efficacy when used in combination with other growth factor inhibitors[33].

Angiogenesis is an important event not only for HCC, but also for cancer growth and metastasis, and occurs because of complex alterations involving promoting factors such as VEGF, angiopoietin, and FGF, and inhibitory factors including thrombospondin and angiostatin, as well as the surrounding tissue. The VEGF family consists of VEGF-A, -B, -C, -D and –E, and placental growth factor (PIGF). The VEGFR family comprises VEGFR-1 (flt-1), VEGFR-2 (flk-1/KDR), and VEGFR-3 (flt-4). VEGF-A binds to VEGFR-1 and -2 and is involved in angiogenesis and the maintenance of mature blood vessels, while VEGF-C and -D mainly bind to VEGFR-3, are involved in lymphangiogenesis[34,35]. VEGF isoforms, such as VFGF₁₂₁ and VEGF₁₆₅, have been identified, and isoform subtypes also exist, such as EGF₁₆₆b. Thus, it is clear that these growth factors do not exhibit angiogenesis-promoting effects alone, and they have attracted attention as new therapeutic targets[36].

HCC typically exhibits active angiogenesis. During the progression from early to well- and moderately-differentiated HCC, angiogenesis increases and cancer cells acquire the ability to invade vessels and metastasize. Scientific and clinical studies have revealed that, during the progression from hepatitis to cirrhosis, angiogenesis and disruption of the vascular architecture are linked to the progression of HCC, and contribute to increased hepatic vascular resistance and portal hypertension, and decreased hepatocyte perfusion[37]. In addition, a meta-analysis has demonstrated that VEGF expression is a prognostic factor in HCC[38].

Phase II studies have been started to test the usefulness of bevacizumab (Avastin^®), which directly targets VEGF, in TACE-treated HCC, and the use of bevacizumab in combination with erlotinib (Tarceva^®), an EGFR tyrosine kinase inhibitor.

Sunitinib (Sutent^®) is a multi-kinase inhibitor that inhibits tyrosine kinases, such as VEGFR-1, -2, -3, PDGFR-α, -β and c-Kit. A phase II study of sunitinib in 37 advanced HCC patients showed that the median progression-free survival (PFS) and median overall survival (OS) were 3.7 and 8 mo, respectively. In that study, adverse events included grade 3/4 thrombocytopenia in 37.8% of patients, neutropenia in 24.3%, asthenia in 13.5%, and hand-foot syndrome in 10.8%[39]. Since sunitinib has a lower IC₅₀ for each target than sorafenib, it is expected to exhibit greater antitumor activity. However, this factor may be responsible for the higher incidence of adverse events with sunitinib. The main evaluation item in the above phase II trial was the response rate, which did not reach the expected value, leading to the conclusion that it was a negative study[40]. In that study, sunitinib was administered at 50 mg/d for four weeks followed by two weeks of rest per cycle[39], whereas Zhu et al[40]. used a dosing schedule of 37.5 mg/d for four weeks followed by two weeks of rest per cycle, and reported that the median PFS and OS were 3.9 and 9.8 mo, respectively. An ongoing global cooperative phase III controlled clinical trial to compare sorafenib and sunitinib head-to-head, and to seek approval for first-line indications for advanced HCC, adopted a sunitinib dosing schedule of 37.5 mg/d. However, in a “Reflection and Reaction” regarding the above trial results, Forner et al[41] cast doubt on whether the drugs at this dose could maintain tolerance and ensure efficacy. Consequently, the trial was terminated on March, 2010 because of the recommendation by data monitoring committee based on interim analysis, showing relatively high toxicity and no superior efficacy to sorafenib.

Brivanib is a kinase inhibitor that selectively inhibits VEGFR-1, -2 and -3, and FGFR-1, -2 and -3. Recent studies suggest that tumor progression following treatment with antiangiogenic agents that target the VEGF signaling pathway alone may result from either evasive or intrinsic resistance[42]. Furthermore, there is strong evidence to support the hypothesis that evasive resistance to anti-VEGF blockade is associated with reactivation of tumor angiogenesis by alternative signaling pathways. One such mechanism of resistance is the activation of the FGF signaling pathway[43,44]. Basic FGF (FGF2) is a potent angiogenic factor. Indeed, expression of FGF2 enhances growth, invasion, and angiogenesis of many tumor types[45,46]. Moreover, recent evidence has shown that FGF is overexpressed and activated in HCC, and that high FGF2 levels may predict a poor clinical outcome among patients with HCC[46].

Considering the proposed importance of FGF signaling in HCC angiogenesis, it is clear that novel antiangiogenic agents that combine inhibition of FGF receptor signaling with inhibition of VEGFR signaling might provide a potential mechanism to overcome anti-VEGF resistance in HCC (Figure 6). With this in mind, it is worthwhile considering the potential future impact of brivanib on the treatment of advanced HCC. Brivanib, a small-molecule tyrosine kinase inhibitor, is the first oral selective dual inhibitor of FGF and VEGF signaling. In multiple preclinical models of human xenograft tumors, including patient-derived HCC xenografts, brivanib has shown potent antitumor activity and no overt toxicity when dosed orally[47,48]. Furthermore, brivanib has demonstrated promising antitumor activity and acceptable tolerability in a phase II, open-label study in patients with unresectable locally advanced or metastatic HCC[49,50]. Crucially, within this trial, brivanib showed activity both as first-line therapy (overall survival: 10 mo) or as second-line therapy in patients who had failed prior antiangiogenic treatment, primarily with sorafenib (overall survival: 9.5 mo)[50]. Of note, the incidence of all-grade hand-foot syndrome was only 8% in this study.

Open in New Tab   Full Size Figure   Download Figure

Figure 6 Brivanib may be effective for the failure or resistance of first line anti-angiogenic therapy for vascular endothelial growth factor. In addition, there is a possibility that anti-FGF agents can be first line anti-angiogenic agents. FGF: Fibroblast growth factor; VEGF: Vascular endothelial growth factor; PDGF: Platelet-derived growth factor.

As for brivanib, an international global phase III clinical trial to compare brivanib and sorafenib head-to-head and to seek approval for first-line therapy for advanced HCC has already been started, and the results are eagerly awaited. Since brivanib targets FGF and VEGF, and is associated with relatively mild adverse effects, a second-line study of brivanib in sorafenib-ineffective and -intolerant patients, and a trial to evaluate the use of brivanib in combination with TACE, are underway. Depending on the results of these trials, indications for use in HCC may be obtained; therefore, positive results are eagerly anticipated.

The results have been reported for a phase II study of brivanib in 55 patients (cohort A) who had not received systemic therapy for curatively unresectable HCC and 46 patients (cohort B) previously treated with angiogenesis inhibitors, such as sorafenib or thalidomide[49]. The median TTP and OS were 2.8 mo and 10 mo, respectively, in cohort A versus 1.4 mo and 9.8 mo, respectively, in cohort B. Adverse events included fatigue (51.5%), diarrhea (41.6%), hypertension (42.6%), anorexia (41.6%), and nausea/vomiting (40.6%/30.7%) in total. Thus, these results demonstrated the efficacy of brivanib as a second-line treatment. The results of three phase III clinical trials, BRISK-PS (sorafenib failure or sorafenib-intolerant patients; brivanib + best supportive care (BSC) vs placebo + BSC), BRISK-FL (advanced HCC; brivanib vs sorafenib), and BRISK-TA (patients with unresectable HCC, brivanib vs placebo as post-TACE adjuvant therapy) are awaited (Table 2).

Linifanib (ABT-869), which strongly inhibits VEGFR and PDGFR, is also under global phase III trial.

In a Japanese phaseI/II trial of TSU-68, an oral molecular inhibitor of VEGFR, PDGFR, and FGFR, to test its safety and efficacy in 35 HCC patients, the response rate was 5.6% (CR, PR, SD, PD and NE in 1, 2, 15, 16 and 1 patients, respectively), and the disease control rate was 51.4%[51]. The global phase III trial of TACE in combination with TSU-68 has just started on January 2011.

In addition, several phaseI/II trials are being conducted to assess kinase inhibitors such as cediranib (AZD2171), which inhibit VEGFR, PDGFR, CSF-1R (cFms), Kit, and Flt3. Furthermore, a phase III global study of axitinib, which is currently being tested in renal cell carcinoma, has also been started as a second line agents on 2011.

EGFR is a member of the HER family, which includes EGFR (erbB1), HER2/neu (erbB3), and HER4 (erb4). All members of this family, except HER3, have an intracellular tyrosine kinase domain, and the binding of a ligand to its extracellular domain triggers signal transduction through the above-described MAPK and PI3K/Akt/mTOR pathways. Thus, these receptors are involved in cell growth, differentiation, survival, and adhesion[52]. EGFR over expression has been reported in many cancers, and in HCC. For example, Buckley et al[53] reported that EGFR, detected by immunohistochemical analysis, was overexpressed in 50 (66%) of 76 HCCs, and that fluorescence in situ hybridization showed extra EGFR gene copies in 17 (45%) of 38 HCCs.

EGFR-targeting drugs include anti-EGFR antibodies, such as cetuximab and panitumumab, and small-molecule inhibitors of EGFR tyrosine kinases, such as gefitinib etc, and have been used widely for the treatment of several cancers other than HCC. Unfortunately, except for phase II trial data, there are little clinical data on the efficacy of these drugs for the treatment of HCC.

Similar to gefitinib (Iressa^®), erlotinib (Tarceva^®) is an oral EGFR tyrosine kinase inhibitor. Philip et al[54] and Yau et al[55] have reported the results of phase II studies of erlotinib in HCC; the median OSs in their studies were 13 and 10.7 mo, respectively. A phase III clinical study (SEARCH study: Sorafenib and Erlotinib, a Randomized Trial Protocol for the Treatment of Patients with Hepatocellular Carcinoma) for sorafenib in combination with erlotinib vs sorafenib plus placebo is ongoing. Since erlotinib is associated with a high incidence of skin rash, dry skin and gastrointestinal toxicity, such as diarrhea, the results of the SEARCH study should be evaluated to assess whether this combination therapy can be used in clinical settings. Thomas et al[56] conducted a phase II clinical study of erlotinib in combination with bevacizumab in 40 advanced HCC patients, and reported promising results; the median PFS and OS were 9 mo and 15.7 mo, respectively. However, they noted frequent treatment-related grade 3/4 toxicities, including fatigue (20%), hypertension (15%), gastrointestinal bleeding (12.5%), wound infection (5%), diarrhea (10%), elevated transaminase levels (10%), and thrombocytopenia (10%), which necessitates further evaluation for drug tolerance. Although a clinical study of erlotinib in combination with bevacizumab (OPTIMOX-3 study) was also conducted in colorectal cancer patients, no tolerance was observed, which led to a change in the protocol[57,58].

After the introduction of a number of molecular-targeted drugs, strategies for the inhibition of similar or different signaling pathways (vertical or horizontal inhibition) with several drugs have been proposed. However, the combined use of molecular-targeted agents has remained largely unsuccessful, including panitumumab in combination with bevacizumab for the treatment of colorectal cancer[59]. Similarly, the results of sorafenib in combination with bevacizumab (vertical inhibition) have been reported[60]. Although some therapeutic responses were obtained, the combination therapy resulted in greater toxicity[60], suggesting the need for detailed evaluation of the dosing regimen.

Lapatinib (Tykerb^®) is a dual inhibitor of EGFR and HER-2/neu, and inhibits tumor growth by downregulating MAPK, AKT, and p70S6 kinase[61]. In Japan, lapatinib is indicated for the treatment of breast cancer. In a phase II clinical trial of lapatinib in 26 patients with unresectable advanced HCC, the median PFS and OS were 1.9 mo and 12.6 mo, respectively, and adverse events included diarrhea (73%), nausea (54%), and skin rash (42%)[62].

Cetuximab (Erbitux^®) is a human/mouse chimeric monoclonal antibody consisting of the variable region of a mouse anti-human EGFR monoclonal antibody and the human immunoglobulin G1 constant region. Cetuximab inhibits the binding of endogenous EGFR ligands, such as EGF and TGFα, to EGFR. In a phase II clinical trial of cetuximab in 30 patients with unresectable or metastatic HCC, the median PFS and OS were 1.4 mo and 9.6 mo, respectively, and treatment-related toxicities included grade 3 hypomagnesemia (3.3%) and grade 1/2 acne-like rash (83.3%), which was observed for the duration of anti-EGFR therapy in that study[63].

The EGFR offers a very interesting therapeutic target. As described above, the use of erlotinib in combination with sorafenib is still in the research stage. However, based on the results of phase II studies, the efficacy of cetuximab or lapatinib as a monotherapy seems to be limited, and the results of further studies evaluating their efficacy in sorafenib-refractory or -intolerant patients are awaited with interest.

Since the hepatocyte growth factor (HGF)/Met pathway is involved in tumor growth, invasion, and angiogenesis in a wide range of neoplasms, HGF and Met have recently attracted attention as therapeutic targets. HGF is a heterodimer consisting of α and β chains bound together by a disulfate bond. The α-chain contains four kringle domains, and the β-chain contains a serine protease-like domain. Met is a receptor tyrosine kinase for the HGF ligand, and contains a semaphorin-like domain. HGF or Met overexpression, and Met gene mutations and duplications, have been reported in various cancers, and abnormalities due to HGF/Met pathway activation have also been noted[64]. These abnormalities activate the downstream signaling cascade, leading to epithelial-mesenchymal transition and increased proliferative, migratory, invasive, and metastatic potentials of cancer cells[64].

HGF/c-MET-targeted drugs, including kinase inhibitors, HGF inhibitors and decoy c-Met receptor molecules are being developed. Of particular interest is ARQ-197, a c-Met receptor tyrosine kinase inhibitor, which is a non-ATP-competitive molecule that binds near the ATP-binding site. A randomized phase II study of ARQ-197 vs placebo is ongoing in patients with unresectable HCC after systemic therapy failure. In addition, the results of a phaseIstudy of ARQ-197 in combination with sorafenib was reported in ASCO 2010 (Abstract 3024).

The IGF/IGFR system is involved in cell growth and the chemotherapeutic response. The ligands IGF-Iand -II bind to their receptors IGF-1R and IGF-2R, and are involved in DNA synthesis and cell growth. Abnormalities in IGF and IGF-1R, or their overexpression, have been reported in various cancers, including HCC. Their associations with disease stage, metastasis, survival[65], and the functions of IGF and IGFR in HCC[66] have been reported.

IGF-targeting drugs are currently being developed, and are mainly anti-IGF-1R antibodies, such as BIIB022, AVE1642, and cixutumumab (IMC-A12). A phase II study of cixutumumab, a phaseIb/II study of sorafenib vs sorafenib plus BIIB022, and phaseI/II studies of AVE1642 as monotherapy or in combination with sorafenib or erlotinib are ongoing.