Angiogenesis and anti-angiogenesis in hepatocellular carcinoma

Cancer Treatment Reviews (2006) 32, 437– 444

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TUMOUR REVIEW

Angiogenesis and anti-angiogenesis in hepatocellular carcinoma Domenico Ribatti a,*, Angelo Vacca b, Beatrice Nico a, Domenico Sansonno b, Franco Dammacco b a

Department of Human Anatomy and Histology, University of Bari Medical School, Piazza Giulio Cesare, 11 Policlinico, 70124 Bari, Italy b Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Received 11 March 2006; revised 1 June 2006; accepted 2 June 2006

KEYWORDS

Summary Experimental and clinical data indicate that in human hepatocellular carcinoma (HCC) tumor progression is associated with angiogenesis and that an increase in microvascular density is associated with a poor prognosis. This review summarizes the literature concerning the relationship between angiogenesis and progression in HCC. It is becoming increasingly evident that agents which interfere with blood vessel formation also block tumor progression. Accordingly, anti-angiogenic tumor therapy has gained much interest in preclinical and clinical assessments. The recent applications of anti-angiogenic agents which interfere or block HCC progression are reviewed. c 2006 Elsevier Ltd. All rights reserved.

Angiogenesis; Anti-angiogenesis; Hepatocellular carcinoma; Tumor progression



Tumor angiogenesis Angiogenesis and the production of angiogenic factors are fundamental for tumor growth, invasion and metastasis.1 New vessels promote growth by conveying oxygen and nutrients and removing catabolites, while endothelial cells secrete growth factors for tumor cells1 and a variety of matrix-degrading proteinases that facilitate tumor inva* Corresponding author. Tel.: + 39 80 5478240; fax: +39 80 5478310. E-mail address: [email protected] (D. Ribatti).



sion.2 An expanding endothelial surface also gives tumor cells more opportunities to enter the circulation and metastasize,3 while their release of anti-angiogenic factors explains the control exerted by primary tumors over metastasis. These observations suggest that tumor angiogenesis is linked to a switch in the equilibrium between positive and negative regulators.4 In normal tissues, vascular quiescence is maintained by the dominant influence of endogenous angiogenesis inhibitors over angiogenic stimuli. Tumor angiogenesis, on the other hand, is induced by increased secretion of angiogenic factors and/or down-regulation of angiogenesis inhibitors.

0305-7372/$ - see front matter c 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ctrv.2006.06.002

438

Angiogenesis in hepatocellular carcinoma There are two types of microvascular structures in the liver: large vessels, such as portal and central venules and hepatic arterioles, lined with continuous endothelial cells, and fenestrated sinusoids lined with discontinuous sinusoidal endothelial cells (SEC).5 The adult liver is highly vascularized. Its cells are flanked by SEC-lined sinusoids and separated from them by the spaces of Disse.6 In liver tumors capillaries are formed and sinusoid fenestration is lost. Stellate cells are regarded as liver-resident pericytes. They may contribute to angiogenesis through mechanisms different from those attributed to pericytes and become activated. Fibrillar extracellular matrix accumulates and hepatocytes lose microvilli. The result is a distorted sinusoidal structure characterized by capillarization and formation of neovessels. A unique proangiogenic factor, namely the recently described AN-GPTL3, a liver-specific secreted factor showing angiogenic properties by binding to avb3 integrin, has been identified.7 Unlike the dual supply of the normal hepatic parenchyma provided by vessels arising from the systemic arterial circulation and the portal venous circulation, an advanced hepatocellular carcinoma (HCC) is abundantly supplied by systemic arteries alone.8 This abundancy facilitates the angiographic diagnosis of malignancy and differentiates non-neoplastic or preneoplastic conditions, such as liver cirrhosis, hepatocellular adenoma, focal nodular hyperplasia and adenomatous hyperplasia. HCC progresses from a small, well-differentiated tumor with no developed blood vessels to a larger and moderately or poorly differentiated form with a characteristic hypervascularity during dedifferentiation process.9–11 Many immunohistochemical and ultrastructural studies have investigated the immunophenotypical and morphological changes in liver sinusoids and SEC during liver carcinogenesis. These changes, known as sinusoidal capillarization, seem to be concomitant with the malignant transformation of precancerous lesions.12 Modifications of the immunophenotype vascular profile consist of abnormal distribution of endothelial markers (factor VIII-related antigen, CD31, CD34 and Ulex europaeus agglutinin-1) and changes in the basement membrane components (laminin and collagen type IV). Angiogenesis in HCC is usually unremarkable, whereas advanced forms are richly supplied with blood vessels.

Endogenous angiogenic stimulators expressed in HCC Vascular endothelial growth factor (VEGF) Since liver cells express VEGF13,14 and endothelial cells express its tyrosine kinase receptors VEGFR-1 and VEGFR2,15,16 it has been suggested that they communicate by means of VEGF signaling: signaling via VEGFR-1 induces SEC to release cytokines that stimulate hepatocyte proliferation; signaling via VEGFR-2 stimulates SEC proliferation.17 VEGF is expressed in HCC.18,19 Mise et al.19 have shown that most HCC samples display a stronger expression of VEGF mRNA. No apparent correlation was observed between the angiogenic gene expression and tumor size or grade.

D. Ribatti et al. Chow et al.20 found that immunoreactivity for VEGF is present in the extracellular matrix of the portal tracts in the normal liver, whereas reactivity in neoplastic tissue may characterize a progression towards higher proliferation in vivo. Yamaguchi et al.18 reported that VEGF protein expression is related to HCC grade: the highest expression was observed in well-differentiated HCC, followed by moderately and poorly differentiated HCC. VEGF expression increases gradually from low-grade dysplastic nodules to high-grade dysplastic nodules to early HCC.21 Its degree correlates with microvascular density and with CD34 staining as a marker of sinusoidal capillarization.21 Tumor expression of VEGF (mRNA and protein expression) significantly correlates with serum VEGF levels in HCC patients.22 Concentration of circulating VEGF increases with advancing HCC stage and is highest in patients with metastasis.23 In 98 patients with resectable HCC, preoperative serum VEGF was a significant and independent predictor of recurrence, disease-free survival and overall survival.24 In a prospective study of 80 patients undergoing transarterial chemoembolization in HCC, pretreatment serum VEGF levels were significantly lower in patients with stable or responsive disease than in those with progressive disease.25 Overexpression of VEGF has been found in HCC compared with cirrhosis or normal liver and is strongly increased in areas directly adjacent to necrotic/hypoxic regions.26

Fibroblast growth factor-2 (FGF-2) A direct correlation between the levels of FGF-2 expression and microvessel density in HCC has been found.27 Moreover, the serum FGF-2 level correlates with clinicopathological features and increased rate of postoperative recurrence.28

Angiogenin Serum concentration and tumoral mRNA expression of angiogenin correlated with microvascular density in a study of 41 HCC patients.29 Angiogenin serum levels significantly decreased after successful TACE or percutaneous ethanol injection. HCC patients with values higher than the mean level had a lower 5-year survival rate.29

Angiopoietins (Ang) Tanaka et al.30 reported that the expression of the Ang-2 gene is up-regulated gene in the hypervascular type of human HCC and that its ectopic expression in non-expressing HCC cells promotes rapid tumor growth and extensive hemorrhage in mice, suggesting that the Ang-Tie system might be involved in angiogenesis in HCC. Mitsuhashi et al.31 investigated the expression of Ang-1, Ang-2, their ligand Tie-2, and VEGF by RT-PCR and immunohistochemical staining in HCC, including adjacent non-tumor liver tissue. VEGF mRNA was significantly upregulated in HCC compared with normal liver tissue, but no differences were found between HCC and adjacent liver tissue. In contrast, Ang-2 mRNA expression in HCC significantly increased when compared with adjacent liver tissue. Ang-1 and Tie-2 expression was not significantly different. Immunohistochemical studies also showed increased Ang-2

Angiogenesis and anti-angiogenesis in hepatocellular carcinoma protein in HCC. The Ang-2/1 mRNA ratios in HCC were closely associated with tumor portal vein invasion, tumor diameter, and the microvascular density. The high Ang-2/ 1 mRNA ratio group showed a significantly poorer prognosis. Sugimachi et al.32 showed that in resected HCC samples, Ang-2 was immunohistochemically detected in more poorly differentiated HCC cells, and Tie-2 was detected in vascular endothelial cells and pericytes. In contrast, Ang-1 expression was observed in normal hepatocytes. Ang-2 has been detected in hepatoma cells at the mRNA level as well as immunohistochemically, whereas its ligand Tie-2 was detected in endothelial cells.33

How different cells and factors contribute to an enhanced angiogenesis in HCC Secretion by HCC cells, infiltrating inflammatory cells and hepatic stellate cells of angiogenic factors, such as VEGF, FGF-2, angiogenin and Angs, promotes the sprouting of new vessels from pre-existing ones. In fact, tumor angiogenesis does not depend on a single molecule, since many angiogenic inducers and inhibitors are simultaneously expressed. Peng et al.34 demonstrated that the counts of tumorassociated macrophages, mast cells and microvessels were significantly higher in metastatic HCC than that in non-metastatic HCC. The infiltrating inflammatory cells secrete several angiogenic factors and may contribute to tumorassociated angiogenesis and there are many reports of associations between macrophage and mast cell infiltration, vascularity and prognosis.35,36 Hepatitis B virus X protein has been shown to increase the transcriptional activity and protein level of hypoxia inducible factor (HIF), a transcriptional factor inducing the expression of several hypoxia response genes, such as VEGF.37 Accordingly, hepatitis B virus promotes angiogenesis during hepatocarcinogenesis.38 Human HCC cells express high levels of matrix metalloproteinases (MMP)39,40 and this overexpression correlates with invasive phenotype.41 MMPs, in turn, promote the release of extracellular matrix-bound or cell-surface-bound cytokines,42 such as VEGF, and further upregulates proangiogenic factors in hepatocarcinogenesis.

Endogenous angiogenic inhibitors expressed in HCC

439 Ishikawa et al.45 stably transfected a vector expressing the angiostatin gene into an HCC cell line to investigate the mechanism of the anti-angiogenic property of angiostatin gene transduction on the expression of VEGF and the effect of transfection on the proliferation and migration of cultured HUVECs in vitro and on the progression of tumors implanted subcutaneously in nude mice. Results showed that expression of angiostatin in HCC cells did not influence the expression of VEGF. However, conditioned media derived from angiostatin-gene-transfected cells suppressed the proliferation and migration of HUVECs. Lastly, suppression of tumor growth was noted in mice implanted with these cells and analysis of the tumor vessel density showed that growth suppression by angiostatin correlated with suppression of vascularity.

Thrombospondin-1 (TSP-1) Kawahara et al.46 reported that the mRNA level of TSP-1 significantly increased in hypovascular cholangiocarcinoma compared to hypervascular HCC, whereas Poon et al.47 showed that TSP-1 expression is associated with invasion and tumor progression in HCC.

Anti-angiogenesis in HCC (Table 1) In 1971, Folkman48 advanced the view that tumor growth depends on angiogenesis and that targeting such angiogenesis could be a new way of preventing tumor progression. Three strategies block tumor growth in experimental models through regression of angiogenesis: vascular targeting, gene therapy and direct inhibition of proliferating and migrating endothelial cells. Alternatively, indirect antiangiogenic drugs prevent the expression or block the activity of tumor proangiogenic factors by interfering with their endothelial cell receptors. Direct angiogenesis inhibitors target the microvascular endothelial cells recruited to the tumor and prevent them from responding to mitogens and motogens. Indirect angiogenesis inhibitors generally prevent the expression of or block the activity of a tumor protein that activates angiogenesis, such as FGF-2 and VEGF, or block the expression of its receptors on endothelial cells. Approximately 75 anti-angiogenic compounds have been developed and are being tested clinically. An overview of ongoing studies on anti-angiogenic treatment is provided by the National Cancer Institute (http://www.cancer.gov).

Endostatin Tyrosine kinase inhibitors Dhar et al.43 measured serum endostatin, VEGF and FGF-2 levels by ELISA in samples from 33 patients who had received no preoperative therapy. They demonstrated a significant decrease in postoperative compared with preoperative serum endostatin and FGF-2 values, and a very strong direct correlation between VEGF and endostatin. Moreover, they found that the serum endostatin was significantly higher in living patients and those with a high level had tended to survive longer. Similarly, Hu et al.44 reported that poorly differentiated hepatoma cell lines exhibited increased endostatin levels compared with the well-differentiated ones.

PTK 787/ZK22254 is a small molecular weight inhibitor of the tyrosine kinase activity of VEGFR-1 and VEGFR-2 and is given orally.49 Preclinical studies have demonstrated its inhibition of tumor vasculature, alone or in combination with chemotherapy or radiotherapy. Liu et al.50 examined the effects of PTK 787 on tumor growth and angiogenesis of HCC xenografts in nude mice. Oral administration significantly reduced tumor volume and microvessel formation, inhibited tumor cell proliferation in a dose-dependent manner and also induced tumor cell apoptosis both in vivo and in vitro. The proapoptotic

440 Table 1

D. Ribatti et al. Antiangiogenic agents applied for treatment of HCC

Agent

Mechanism of action

Stage

References

Bevacizumab Thalidomide IFN-a IL-12 PTK 787 Gefitinib TNP-470 Batimastat (BB-94) Endostatin

Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition Inhibition

Phase II Phase II Phase II Murine model Murine model Murine model Murine model Murine model Phase I

Schwartz et al.56,57 Lin et al.65 Patt et al.66 Peron et al.77 Liu et al.50 Matsuo et al.53 Kin et al.81 Bu et al.82 Eder Jr., et al.85

of of of of of of of of of

VEGF endothelial cell proliferation endothelial cell migration angiogenesis tyrosine kinase EGFR angiogenesis MMP tumor cell growth

response was associated with down-regulation of Bcl-2 and Bcl-xL expression and induction of cleavage of caspase 3. In addition, PTK 787 induced growth arrest in HCC cells. This was associated with G1 arrest and partial G2-M block. Raskpof et al.51 used a recombinant adenoviral vector encoding a soluble dominant negative fragment of VEGFR2, Ads Flk-1, to control murine orthotopic and metastatic hepatomas. Systemic treatment of tumor-bearing mice inhibited tumor growth by 84% compared with the corresponding control group. Numerous reports have described overexpression of epidermal growth factor receptor (EGFR) in epithelial tumors and substantiated the view that deregulated EGFR signaling has an important role in human cancer. EGFR inhibitors are currently under clinical development. Many display antiangiogenic activity, at least in preclinical models, and this could partly explain their clinical efficacy.52 In a murine HCC model, oral Gefitinib (ZD 1839 or Iressa), a potent EGFR inhibitor, inhibited the growth of implanted HCC and intrahepatic metastasis by approximately 50% and prevented EGF-induced chemotactic migration of HCC cells and production of active MMP-9 in vitro.53

Thalidomide Thalidomide is a compound with anti-angiogenic, immunomodulatory and antitumor effects. Its most promising clinical results have been obtained in multiple myeloma,59 whereas it has proved poorly effective in recurrent glioma, breast cancer, melanoma, renal and ovarian cancer and hormone-refractory prostate cancer.60–62 Hsu et al.63 reported the results of a trial with 68 patients with unresectable and non-embolizable HCC, treated with thalidomide. One complete and three partial responses were seen, with a response rate of 6.3%. Wang et al.64 treated 99 patients and obtained six partial responses. Lin et al.65 treated 27 patients with unresectable HCC. One patient showed near complete recovery of a-fetoprotein levels and a partial radiographic response; another two patients had stable disease. The overall median survival was 123 days. Finally, Patt et al.66 treated 37 patients with thalidomide and showed that one patient had a partial response, one a minor response and 10 a stable disease. The overall median survival was 6.8 months.

Interferon alpha (IFN-a) Bevacizumab (Avastin) Bevacizumab is a humanized murine anti-VEGF monoclonal antibody, which recognizes all VEGF isoforms without cross-reacting with other growth factors, displaying potent antitumor activity in experimental models.54 First-line use of bevacizumab with irinotecan, 5-fluorouracil and leucovorin produced significantly improvements in response rate, duration of response and survival in a phase III study of patients with metastatic colorectal cancer.55 Bevacizumab has been tested in a phase II study in patients with unresectable non-metastatic HCC and without main portal invasion.56,57 Bevacizumab is safe and potentially efficacious in localized HCC in patients with adequate liver function and results suggest significant disease-modifying effect, including six month stability in patients with prior rapid growth.56,57 More recently, a phase II study undertaken to examine the efficacy and safety profiles of combining bevacizumab with the combination of gemcitabine and oxaplatin (GEMOX) in patients with unresectable or metastatic HCC, has demonstrated a moderate antitumor activity of this combination.58

IFN-a was first shown to inhibit endothelial cell migration by Brouty-Boye and Zetter.67 This led to its subsequent characterization as an angiogenesis inhibitor. Its anti-angiogenic effects in humans are illustrated by its efficacy in the treatment of pediatric hemangiomas,68 which express high levels of FGF-2 as their major angiogenic mediator. Metastasis and recurrence of human HCC after resection were inhibited by IFN-a in a study with nude mice showing decreased VEGF serum levels and decreased tumoral microvascular density, but not FGF-2 levels.69 Shiratori et al.70 treated with IFN-a patients with compensated cirrhosis, three or fewer nodules of HCC and low HCV RNA loads with complete ablation of lesions by percutaneous ethanol injection therapy. Of the 49 patients treated, 21 showed a sustained biochemical response and 14 showed a sustained virologic response. A clinical prospective randomized controlled study found that IFN-a prevented HCC recurrence reduced after medical ablation therapy for primary tumors.71 In a phase II trial, 34 patients with HCC and 9 with fibrolamellar HCC were treated with subcutaneous recombinant IFN-a2b and systemic

Angiogenesis and anti-angiogenesis in hepatocellular carcinoma continuous 5-fluorouracil.72 A partial response was observed in 14% of those with HCC and 62.5% of those with fibrolamellar HCC.

441 Rats with deficient-diet-induced HCC treated with TNP470 developed fewer and smaller tumors than control animals. This was accompanied by a reduction of tumor vascularity.81

Interleukin-12 (IL-12) TIMPs Il-12 blocks corneal angiogenesis in normal and immunodeficient mice, suggesting that the presence of immunocompetent T-cells is not a prerequisite for its anti-angiogenic activity.73 Il-12 acts via the release of IFN-b and consequently interferon-inducible protein 10 (IP-10), a member of the CXC chemokine family, which can itself block the activity of FGF-2.74 In addition, down-regulation of MMP-9 and increase of tissue inhibitor of metalloproteinase (TIMP-1) may also contribute to the angioinhibitory effects of IL-12.75 In a rat model of orthotopic HCC, adenovirus-mediated gene transfer of IL-12 inhibited tumor growth in a dosedependent manner.76 Similar results were observed in a murine model with either tumor cells expressing IL-12 or injection of the tumor with fibroblasts secreting IL-12.77

TNP-470 TNP-470 (or AGM-1470) is a synthetic analog of fumagillin, a natural angiogenesis inhibitor. It acts by inhibiting the phosphorylation of methionine aminopeptidase-2, a cytoplasmic enzyme of endothelial cells, which is overexpressed in tumor cells. TNP-470 has been applied in clinical trials in patients with carcinoma of the cervix metastasized to the lung78 and renal cell carcinoma.79 Combination of TNP470 with conventional cytotoxic agents, such as cisplatin, paclitaxel or cyclophosphamide, significantly improve their antitumor efficacy.80

Endostatin In preclinical studies in mice endostatin has been found to inhibit the growth of metastatic and primary liver tumors without drug resistance or toxicity.83,84 A phase I clinical trial of recombinant human endostatin given in 15 patients with refractory solid tumors showed no significant toxicities and evidence of clinical benefit in three patients.85

The winning strategy The complex relationship between angiogenic cascade and anti-angiogenic agents in the tumor vascular phase, as well as identification and characterization of angiogenesis inhibitors, have indicated that anti-angiogenesis can be considered as a strategy for the adjuvant therapy of HCC (Fig. 1).

Cirrhotic liver

Normal liver

Sinusoid capillaries

MMP inhibitors have been extensively assessed in a broad range of malignant diseases because of their ability to inhibit MMP-2 and MMP-9, which are thought to be important for an early stage of angiogenesis. Because they inhibit MMP, it was believed they could inhibit tumor growth, invasion, angiogenesis and metastasis. Batimastat (BB-94), a synthetic inhibitor of MMP, inhibited primary tumor growth, local invasion and lung metastasis, and also prolonged survival in an orthotopic metastatic human HCC nude mice model.82

Extracellular matrix

HCC

Fibrosis with laminin and collagen type IV

Capillaries stained with FVIII-RA, CD31, CD34

Tumor nodule with neovessels and fibrosis

Inducers VEGF FGF-2 Angiogenin Angiopoietins

Figure 1

Endogenous inhibitors

Endostatin Thrombospondin-1

Major angiogenesis inducers and endogenous angiogenesis inhibitors in hepatocellular carcinoma (HCC).

442 The results of preclinical studies with anti-angiogenic agents had been very promising showing partly or complete tumor regression without any drug resistance. However, many of these results could not be confirmed in clinical trials. In fact, objective tumor responses were achieved only in some cases. Especially, the minimal antitumoral effects of endostatin in doses equivalent to those used in animal models has been disappointing. VEGF receptors tyrosine kinase inhibitors or thalidomide might actually the most promising substances (Table 1). The advantage of thalidomide is that it is administered orally and, in general, is well tolerated, especially in patients without cirrhosis. Conventional cytotoxic chemotherapeutic drugs have been used at low and non-cytotoxic concentrations as anti-angiogenic. Anti-endothelial effects have been demonstrated in vitro and in vivo for several cytostatic agents. The presence of dividing endothelial cells in newly forming tumor blood vessels should render such vessels, in contrast to the mature vessels found in normal adult tissues, sensitive to the cytotoxic effects of such drugs. The use of low doses in ‘‘metronomic’’ chemotherapy (namely, very frequent or continuous low-dose chemotherapy) as anti-angiogenic targeting strategy, seems particularly effective against drug-resistant tumors, especially when it is combined with a second anti-angiogenic drug.86 Although there are no studies of metronomic chemotherapy in patients with HCC, this modality might be useful in patients with diminished hepatic functional reserve and a reduced capacity to metabolize and excrete chemotherapeutic drugs. There is accumulating evidences that a number of antiangiogenic substances can lose their activity over time. This could be caused by several possible mechanisms, e.g. an overexpression of other angiogenic factors that may antagonize the function of the anti-angiogenic agents if only one of such factors is the target for an anti-angiogenic therapy. For example, blockade of the VEGF pathway seems too narrow as an approach to inhibit angiogenesis, because a tumor may use a compensatory pathway. For this reason, the combined application of angiogenesis inhibitors might be a promising strategy. Inhibition of various receptor/ligand systems could be effective due to the different mechanism of action within the angiogenesis process and combination of inhibitors with different mechanisms of action attacking different biochemical pathways should shut down the multifactor stimulated cascade of biochemical angiogenic processes.

Acknowledgement Supported by Associazione Italiana per la Ricerca sul Cancro AIRC (Regional Funds) Milan, Fondazione Italiana per la Lotta al Neuroblastoma, Genoa, MIUR (FIRB2001 and PRIN2005) Rome, Italy.

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