Immune Escape in Hepatocellular Cancer: Is a Good Offense the Best Defense?
Hepatology, August 1999, p. 576-578, Vol. 30, No. 2
Our immune system, charged with the function of cancer surveillance, has the complex but efficient capability to clear genetically altered cells that have undergone malignant transformation. Tumor cells must develop strategies to avoid clearance by the immune system to survive, expand their populations, and metastasize. The process by which unwanted tumor cells are cleared involves recognition of the altered nature of the cell by the immune system followed by its effective killing and elimination. Thus, tumor cells may escape immune clearance by altering immune recognition or by modulation of the cytotoxic response.
The recent identification of the Fas receptor (Fas, APO-1/CD95) and its ligand (FasL, CD95L) as a major regulator of both apoptosis and immune function has provided insight into an attractive mechanism of tumor escape from immune clearance. Fas and FasL are transmembrane proteins of the tumor necrosis factor family of receptors and ligands.1 Engagement of Fas by FasL triggers a cascade of well-characterized intracellular signaling events involving homophilic associations of the intracellular death domain, formation of the death-inducing signaling complex, and the recruitment and activation of a cascade of effector caspase proteases. These signaling events culminate in cell death by apoptosis. One of the main mechanisms by which immune effector cells kill is by induction of apoptosis by Fas/FasL interactions. The Fas/FasL system also plays an important physiological role in the termination of the immune response. Fas is induced on activated T lymphocytes and targets them for subsequent elimination by FasL-expressing cells. This limits clonal expansion of activated lymphocytes. In addition, the elimination of Fas-expressing leukocytes also serves to prevent inflammation at sites of immune privilege such as the eye, brain, and testis, which constitutively express FasL.2
Given the importance of the Fas/FasL system as a major mechanism by which T lymphocytes kill tumor cells, it is not surprising that tumor cells can use this system to evade immune recognition. The strategies include both defensive measures such as acquiring resistance to Fas-mediated apoptosis, and offensive measures such as targeted, FasL-mediated, destruction of activated lymphocytes. Indeed, a wide range of tumor-derived cell lines or freshly isolated umor cells exhibit decreased expression of Fas. Tumor cells may acquire resistance to Fas-mediated apoptosis by down-regulating Fas expression, secreting decoy receptors to block FasL on activated T lymphocytes, or by altering intracellular mechanisms mediating Fas-induced apoptosis. Another strategy that has been used by some tumors involves de novo expression of FasL on the cell surface. When these tumor cells encounter activated T cells bearing Fas, FasL/Fas interactions induce apoptosis of the T cell thereby terminating the immune response and providing immune privilege to tumors.3 This strategy, eloquently termed the Fas counterattack, is also of great potential importance in the maintenance of immune privilege, acquisition of tolerance, and allograft protection after transplantation.4 However, direct evidence or definitive proof of the role played by the Fas counterattack in vivo as a mechanism of tumor immune escape is lacking. Observations from some in vivo experimental models suggest antitumoral or proinflammatory properties of FasL expression, thereby undermining the potential role of this strategy in tumor immune escape. Under certain circumstances, expression of FasL results in tumor removal, and overexpression of FasL results in an intense inflammatory response in several experimental models.5-7
In this issue of HEPATOLOGY, Nagao et al. report a detailed assessment of alterations in membrane-bound and soluble Fas andFasL in human hepatocellular carcinoma and correlate these changes with clinical and pathological features of the tumors.8 Although nonmalignant hepatocytes constitutively express Fas, a loss of expression has been identified in malignant hepatocytes by several investigators.9-13 Furthermore, in a recent study, Ito et al. reported an association between loss of Fas expression and the degree of differentiation in hepatocellular cancer.13 These observations were confirmed in the current study, which also showed an association between loss of Fas expression and disease-free survival after resection. Although the expression of FasL was present in some tumors, a multivariate analysis identified the loss of Fas but not gain of FasL expression as a critical factor in determining intrahepatic tumor spread. The observations by Nagao et al. suggest that immune escape in hepatocellular cancer is mediated predominantly via the loss of Fas expression, not expression of FasL. Although Strand et al.9 have shown that expression of FasL in hepatocellular cell lines can induce Fas-mediated apoptosis in cocultured lymphocytes in vitro, the relevance of this as a mechanism of tumor immune escape in vivo is unknown. Fas-positive tumor-infiltrating lymphocytes, for example, are rarely observed in histopathological analysis of human hepatocellular cancer. Furthermore, the expression of FasL by tumor cells might result in autocrine or juxtacrine cell apoptosis unless the tumor and adjacent nonmalignant cells had previously acquired resistance to Fas. The acquisition of Fas resistance, such as by decreased expression of Fas, would thus appear to be an important early feature in hepatocarcinogenesis, correlating with degree of differentiation and with intrahepatic spread. These observations are at odds with reports from other solid tumors such as colon and esophageal cancer in which FasL expression (in the presence or absence of Fas expression) appears to play a more prominent role in tumor immune escape by presumably inducing lymphocyte apoptosis.3,14-16 Liver metastases from colorectal tumors show an increased FasL expression (when compared with their primary tumors) along with a decrease in the number of tumor infiltrating lymphocytes suggesting that expression of FasL provides a growth advantage to metastasizing colorectal tumor cells and facilitates growth in the liver.17-19 Whether a similar mechanism holds for other tumors that metastasize in the liver is not yet known. The differences between primary and malignant liver tumors may be due to a unique role played by the Fas system in the liver. The liver constitutively expresses Fas and is exquisitely sensitive to Fas-mediated apoptosis. Thus, Fas may serve an undetermined physiological function other than merely targeting cells for apoptosis. Up-regulation of FasL is seen in several liver diseases such as alcohol-induced hepatitis, Wilson’s disease, and Hepatitis B, and may be associated with hepatic inflammation.20,21 FasL expression by malignant hepatocytes may potentially be counterproductive, enhancing inflammation and facilitating tumor cell destruction.
An alternative mechanism of Fas resistance involves the use of an antagonistic decoy protein such as soluble Fas (sFas), which is similar to Fas, except that it lacks the transmembrane domain. Elaboration of sFas by tumor cells may contribute to resistance to Fas-mediated apoptosis. Elevated sFas levels in serum have been observed in patients with hepatocellular cancer, indicating that the use of decoy receptors may also play a role in liver cancer.8,22 Several other mechanisms for acquiring Fas resistance have been postulated. Disruption of intracellular signaling by structural alterations or functional inactivation of signaling proteins may alter susceptibility to Fas-mediated apoptosis.23 Some cancer cells, including hepatocellular cancer cells, express high levels of proteins such as Fas-associated phosphatase-1, which interact negatively with the Fas receptor.12 The Fas signal may be abrogated by the recruitment of some proteins such as FLIP (FLICE/caspase 8 inhibitory protein) inhibitor protein. Members of the Bcl-2 family of apoptosis regulators may also modulate Fas-mediated apoptosis in some circumstances. Finally, mutations in oncogenes and tumor suppressor genes such as p53 may also interfere with Fas signaling.
Several recently identified members of the tumor necrosis factor receptor family contain a death domain with homology to that of Fas. These death receptors and their ligands may resemble Fas, although their involvement in immune escape or tumor formation remains unknown.24 Of these, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors share the greatest homology to Fas. Many tumor cell lines are sensitive to TRAIL, whereas normal tissues are not. Although TRAIL receptors 1-4 are expressed on both Hep 3B and Hep G2 cells, only the latter are sensitive to TRAIL-induced apoptosis.25 Thus, resistance to apoptosis is more likely to be mediated by intracellular signaling events than by alterations in receptor expression or the presence of decoy receptors. The relevance of TRAIL and other death-receptor systems to hepatocellular cancer has yet to be elucidated.
The emerging experimental and clinicopathological evidence indicates an important role of defensive strategies aimed at avoiding Fas-mediated apoptosis as a means of immune escape in hepatocellular cancer. Nevertheless, much remains to be learned. Immune-mediated tumor killing can occur by secretion of perforin and granzymes, and this mechanism is also important in tumor immune surveillance.26 As apoptosis can be triggered in several ways, including Fas, perforin/granzyme, or death receptors such as TRAIL, a more efficient approach to immune escape would be for the cell to acquire defects in intracellular apoptotic pathways. Our knowledge of Fas (and other death receptors) is expanding. This will hopefully prompt study of the role and relevance of perturbations in these pathways during immune escape and tumor formation in the liver. Translating this knowledge to clinical practice may eventually prove valuable in developing rational immune-based therapies for hepatocellular cancer.
|Tushar Patel, M.D.
Division of Gastroenterology
Scott and White Clinic and Hospital
Texas A & M University System Health Science Center College of Medicine
Abbreviations: FasL, Fas ligand; sFas, soluble Fas; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.
Received May 26, 1999; accepted June 4, 1999.
From the Division of Gastroenterology, Scott and White Clinic and Hospital, Texas A&M University System Health Science Center College of Medicine, Temple, TX.
Supported by the Scott Sherwood and Brindley Foundation, and grant DK02678 from the National Institutes of Health.
Address reprint requests to: Tushar Patel, M.D., Division of Gastroenterology, Scott and White Clinic, 2401 South 31st St., Temple, TX 76508. E-mail: firstname.lastname@example.org; fax: 254-724-8276.
|1.||Nagata S, Golstein P. The Fas death factor. Science 1995;267:1449-1456 [ Link previously at www.ncbi.nlm.nih.gov ].|
|2.||Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995;270:1189-1192[Abstract].|
|3.||O’Connell J, O’Sullivan GC, Collins JK, Shanahan F. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med 1996;184:1075-1082 [ Link previously at www.ncbi.nlm.nih.gov ].|
|4.||Li XK, Okuyama T, Tamura A, Enosawa S, Kaneda Y, Takahara S, Funashima N, et al. Prolonged survival of rat liver allografts transfected with Fas-ligand expressing plasmid. Transplantation 1998;66:1416-1423 [ Link previously at www.ncbi.nlm.nih.gov ].|
|5.||Arai H, Gordon D, Nabel EG, Nabel GJ. Gene transfer of Fas ligand induces tumor regression in vivo. Proc Natl Acad Sci U S A 1997;94:13862-13867[Abstract/Full Text].|
|6.||Chen JJ, Sun Y, Nabel GJ. Regulation of the proinflammatory effects of Fas ligand (CD95L). Science 1998;282:1714-1717[Abstract/Full Text].|
|7.||Hug H. Fas-mediated apoptosis in tumor formation and defense. Biol Chem 1997;378:1405-1412 [ Link previously at www.ncbi.nlm.nih.gov ].|
|8.||Nagao M, Nakajima Y, Hisanaga M, Kayagaki N, Kanehiro H, Aomatsu Y, Yagita H, et al. The alteration of Fas receptor and ligand system in hepatocellular carcinomas. HEPATOLOGY 1999;30:413-421.|
|9.||Strand S, Hofmann WJ, Hug H, Muller M, Otto G, Strand D, Marian S, et al. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cellsa mechanism of immune evasion? Nat Med 1996;2:1361-1366 [ Link previously at www.ncbi.nlm.nih.gov ].|
|10.||Higaki K, Yano H, Kojiro M. Fas antigen expression and its relationship with apoptosis in human hepatocellular carcinoma and noncancerous tissues. Am J Pathol 1996;149:429-437 [ Link previously at www.ncbi.nlm.nih.gov ].|
|11.||Kubo K, Matsuzaki Y, Okazaki M, Kato A, Kobayashi N, Okita K. The Fas system is not significantly involved in apoptosis in human hepatocellular carcinoma. Liver 1998;18:117-123 [ Link previously at www.ncbi.nlm.nih.gov ].|
|12.||Shin E-C, Shin J-S, Park JH, Kim J-J, Kim H, Kim SJ. Expression of Fas-related genes in human hepatocellular carcinomas. Cancer Lett 1998;134:155-162 [ Link previously at www.ncbi.nlm.nih.gov ].|
|13.||Ito Y, Takeda T, Umeshita K, Sakon M, Wakasa K, Matsuura N, Monden N. Fas antigen expression in hepatocellular carcinoma tissues. Oncology Reports 1998;5:41-44 [ Link previously at www.ncbi.nlm.nih.gov ].|
|14.||Bennett MW, O’Connell J, O’Sullivan GC, Brady C, Roche D, Collins JK, Shanahan F. The Fas counterattack in vivo: apoptotic depletion of tumor-infiltrating lymphocytes associated with Fas ligand expression by human esophageal carcinoma. J Immunol 1998;160:5669-5675 [ Link previously at www.ncbi.nlm.nih.gov ].|
|15.||Bennett MW, O’Connell J, O’Sullivan GC, Roche D, Brady C, Kelly J, Collins J, et al. Expression of Fas Ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer. Gut 1999;44:156-162[Abstract/Full Text].|
|16.||O’Connell J, Bennett MW, O’Sullivan GC, Roche D, Kelly J, Collins JK, Shanahan F. Fas ligand expression in primary colon adenocarcinomas: Evidence that the Fas counterattack is a prevalent mechanism of immune evasion 1998;186:240-246 [ Link previously at www.ncbi.nlm.nih.gov ].|
|17.||Shiraki K, Tsuji N, Shioda T, Isselbacher KJ, Takahashi H. Expression of Fas ligand in liver metastases of human colonic adenocarcinomas. Proc Natl Acad Sci U S A 1997;94:6420-6425[Abstract/Full Text].|
|18.||Yoong KF, Afford SC, Randhawa S, Huscher SG, Adams DH. Fas/Fas ligand interaction in human colorectal hepatic metastases. Am J Pathol 1999;154:693-703[Abstract/Full Text].|
|19.||Mann B, Gratchev A, Bohm C, Hanski ML, Foss HD, Demel G, Trojanek B, et al. FasL is more frequently expressed in liver metastases of colorectal cancer than in matched primary carcinomas. Br J Cancer 1999;79:1262-1269 [ Link previously at www.ncbi.nlm.nih.gov ].|
|20.||Galle PR, Hofmann WJ, Walczak H, Schaller H, Otto G, Stremmel W, Krammer P, et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med. 1995;182:1223-1230 [ Link previously at www.ncbi.nlm.nih.gov ].|
|21.||Luo K-X, Zhu YF, Zhang LX, He HT, Wang XS, Zhang L. In situ investigation of Fas/FasL expression in chronic Hepatitis B infection and related liver diseases. J Viral Hepat 1997;4:303-307 [ Link previously at www.ncbi.nlm.nih.gov ].|
|22.||Jodo S, Kobayashi S, Nakajima Y, Matsunaga T, Nakayama N, Ogura N, Kayagaki N, et al. Elevated serum levels of soluble Fas/APO-1 (CD95) in patients with hepatocellular carcinoma. Clin Exp Immunol 1998;112:166-171 [ Link previously at www.ncbi.nlm.nih.gov ].|
|23.||O’Connell J, Bennett MW, O’Sullivan GC, Collins JK, Shanahan F. The Fas counterattack: cancer as a site of immune privilege. Immunol Today 1999;20:46-52 [ Link previously at www.ncbi.nlm.nih.gov ].|
|24.||Faubion WA, Gores GJ. Death receptors in liver biology and pathobiology. HEPATOLOGY 1999;29:1-4.|
|25.||Ganten T, Mueller M, Stremmel W, Krammer PH, Walczak H. Drug Modulated sensitivity of human hepatoma cell lines for Trail-induced apoptosis [Abstract]. Gastroenterology 1999;116:A1214.|
|26.||van den Broek ME, Kagi D, Ossendorp F, Toes R, Vamvakas S, Lutz WK, Melief C, et al. Decreased tumor surveillance in perforin-deficient mice. J Exp Med 1996;184:1781-1790 [ Link previously at www.ncbi.nlm.nih.gov ].|