The Universal Character of the Tumor-Associated Antigen Survivin

The function of survivin. Most malignancies are characterized by defects in apoptosis signaling. Among the regulators of apoptosis implicated in cancer cell survival, Bcl-2 proteins are thought to regulate the mitochondria permeability transition by inhibiting or promoting cytochrome c release, whereas members of the inhibitors of apoptosis (IAP) gene family function as endogenous inhibitors of caspases (1). Eight human IAP family members have been identified thus far. They are c-IAP1, c-IAP2, XIAP, NAIP, survivin, apollon, ML-IAP/livin, and ILP-2 (2). Survivin has attracted attention as a unique member of the IAP gene family with a potential dual role in apoptosis inhibition and regulation of mitosis (3).

The role of survivin in the regulation of mitosis has recently become better understood, and linked to multiple spindle microtubule functions and mitotic checkpoints (4, 5). Thus, in normal cells, survivin is preferentially expressed at mitosis in a cell cycle–dependent manner and is physically associated with the mitotic apparatus. Survivin is essential for the proper completion of various stages of cell division, from centrosomal functions to proper kinetochore attachment to spindle formation, potentially via the regulation of microtubule dynamics/stability.

In contrast, despite extensive experimental evidence in vitro, and in transgenic animals in vivo (3), the precise mechanism(s) by which survivin interferes with apoptosis has not been elucidated. Survivin counteracts cell death by interfering with caspase-9 processing (6), the upstream initiation of the intrinsic (mitochondrial) pathway of apoptosis. Furthermore, cytoprotection by survivin is more selective than that by other IAPs, and is specifically targeted at the initiation of mitochondrial apoptosis to prevent caspase-9 activation (1).

Expression of survivin. Survivin is abundantly and ubiquitously present in development, is undetectable in most adult tissues, and is prominently re-expressed in virtually every human cancer. Thus, one of the most significant features of survivin is its preferential expression in tumor versus normal tissues. Even though a few normal cells do express survivin, e.g., thymocytes, CD34+ bone marrow–derived stem cells, and basal colonic epithelial cells (3), under physiologic conditions, survivin is undetectable in most terminally differentiated normal tissues (7). In normal cells, survivin expression at G₂-M phase is followed by a rapid decline at G₁ phase. However, it has been suggested that the survivin gene might be globally deregulated in transformed cells, leading to overexpression at all phases of the cell cycle, not just during the G₂-M phase (3). A global deregulation of the survivin gene mediated by oncogenes, including signal transducers and activators of transcription 3, E2F, or mutated Ras, or by loss of tumor suppressors, like p53 or the adenomatous polyposis coli protein, accounts for the selective expression of survivin in cancer (1).

Survivin is overexpressed in almost all cancers including lung, colon, breast, pancreas, stomach, liver, ovary and prostate cancer, as well as in melanoma and hematopoietic malignancies (8–10). Recently, it was reported that expression of survivin and the inhibitory T cell ligand B7-H1 could be used to predict clear cell renal cell carcinoma tumor aggressiveness (11). Data from a large analysis of human transcripts revealed survivin as the fourth most highly expressed protein in human cancer tissue compared with normal tissue (12). The extremely high expression of survivin in cancer carries prognostic and predictive implications and, through molecular profiling, is consistently associated with advanced disease, high grade, abbreviated survival, resistance to therapy, and accelerated recurrences. Clinical exploitation of survivin for cancer molecular diagnostics is under way, with its inclusion as 1 of 16 genes predictive of recurrences in breast cancer (13) and as a urine biomarker in bladder cancer (14).

Survivin in therapy. Essentially all cytotoxic anticancer drugs, e.g., microtubule-binding drugs, DNA-damaging agents, and nucleosides currently in clinical use, induce the apoptosis of malignant cells. Up-regulation of IAP proteins plays a vital role in resistance to chemotherapy and radiotherapy (15). Thus, overexpression of survivin in cancer cells is associated with decreased overall survival (16–18), an increased rate of recurrences, and a reduced apoptotic index of neoplastic cells in vivo (3). Although conventional drugs are important weapons in the treatment of cancer, new classes of targeted therapeutics are emerging based on strategies that rely on a deeper understanding of the molecular mechanisms that underlie the phenomenon of apoptosis. In view of this, strategies aimed at inhibiting the expression or function of antiapoptotic proteins has gained considerable attention (19, 20). Thus, although survivin overexpression leads to increased resistance to apoptotic stimuli such as chemotherapeutic agents or ionizing radiation therapy, antisense-mediated silencing of survivin sensitizes the cells to these interventions (21). Supported by a favorable safety profile, the original survivin antisense oligonucleotide has now completed a phase I trial in patients with advanced cancers, and a phase II trial has been announced. A parallel strategy to suppress survivin levels in tumor cells involved RNA interference (22) or hammerhead ribozymes (23). These reagents produced a phenotype similar to antisense and passed proof-of-concept with inhibition of tumor growth in xenograft models. Another strategy is the use of synthetic peptides that compete with caspases for binding to IAPs, which have been shown to sensitize tumor cell lines to apoptosis induced by cytotoxic anticancer drugs (24). Several clinical trials using these reagents are under way. One of these molecules, tetra-O-methyl nordihydroguaiaretic acid was shown to function by suppressing Sp1-dependent survivin gene expression, resulting in the concomitant activation of mitochondrial apoptosis in tumor cells (25).

In addition to the abovementioned means of targeting survivin, immunomediated tumor destruction is emerging as an interesting modality to treat patients with cancer. A significant effort in this field is aimed at the elucidation of tumor-associated antigens. Accordingly, down-regulation of survivin would severely inflict the survival capacity of tumor cells, which highlights this protein as a prime target candidate for therapeutic vaccinations against cancer because it is not subject to immune selection, i.e., the selection of cancer cells not expressing the vaccination target. In this regard, several recent reports have—as we will see below—shown that survivin is among the tumor antigens that serve as targets for immunomediated tumor destruction in patients with cancer.

Presentation of survivin-derived peptides by cancer cells to the immune system. Cellular immunity is largely based on T lymphocytes. A T cell expresses a unique antigen-binding molecule called the T-cell receptor on the cell surface. In contrast to membrane-bound antibodies on B cells, which can recognize antigen alone, the vast majority of T-cell receptors recognize a complex ligand, comprising an antigenic peptide bound to MHC-derived molecules (26). The formation of peptide-MHC complexes requires that a protein antigen is degraded by a sequence of events called antigen processing. CTLs recognize antigenic peptides in the context of MHC class I molecules. The generation of mature, peptide-loaded class I molecules at the cell surface requires the coordination of three essential processes: first, the degradation of proteins to peptides in the cytosol; second, the translocation of peptides across the endoplasmic reticulum membrane; and third, the assembly of MHC class I/peptide heterotrimers and their transport to the cell surface (Fig. 1; ref. 27).

CTLs have a vital function in monitoring the cells of the body and eliminating those cells displaying the respective antigen. CTL are thought to provide the major part of the immune surveillance against tumors by virtue of their ability to detect quantitative and qualitative antigenic differences in transformed cells (28). Oncogenic alterations result in an altered protein repertoire inside the cell, e.g., high expression of survivin. Subsequently, class I MHC molecules sample survivin peptides derived from protein degradation inside the cell and present these at the cell surface (Fig. 1). When a T cell encounters antigen in the context of an HLA molecule, it undergoes clonal expansion and differentiates into various effector as well as memory T cells.

Survivin-specific immune responses. In the February 2007 issue of Clinical Cancer Research, Grube and colleagues very convincingly describe that CD8+ T cells react against the survivin antigen in patients with multiple myeloma (29). Likewise, Coughlin et al. recently reported that children with high-risk neuroblastoma harbor robust cellular immune responses to survivin at the time of diagnosis (30). Both studies underline the generality of the tumor antigen survivin. In this regard, spontaneous anti-survivin T-cell reactivity has previously been described in cancer patients suffering from breast cancer, colon cancer, lymphoma, leukemia, and melanoma (31–36).

To explore the lytic capacity of survivin-specific T cells ex vivo, we isolated such cells by means of magnetic beads coated with MHC/peptide complexes. As predicted, these survivin-reactive T cells were capable of lysing HLA-matched tumor cells of different tissue origin, including breast cancer cells and melanoma (32). These observations were confirmed by Schmidt et al. who showed that survivin-specific CTL generated from healthy donors or patients with leukemia elicited cytolytic activity against tumor cells endogenously expressing the survivin protein, including renal cell carcinomas, breast cancer, colon cancer, melanoma, and multiple myeloma cell lines, as well as primary malignant cells from patients with different leukemias (37). Coughlin et al. used CD40-activated B cells transfected with whole tumor RNA, which induced survivin-specific T cells in vitro that lysed HLA-matched neuroblastoma cells but not autologous benign cells (38). Hence, survivin is among the targeted antigens in vaccination strategies using whole tumor cells as an antigen source.

There is consensus that the induction of efficient and powerful T cell responses requires proper activation of the T cell. However, it is equally important that the T cells acquire the ability to home to the site of action. In situ immunohistochemistry using multimerized HLA/survivin-peptide complexes disclosed that survivin-specific CTL could readily be detected in the tumor microenvironment, both in primary tumors and metastases of patients with melanoma as well as in breast cancer lesions (32, 33). The combined detection of survivin-specific T cells in blood and tumor lesions indicates that these cells are capable of circulating and homing to the effector site. This is a significant finding because several clinical reports have indicated the existence of a functional dissociation between local and systemic anti-melanoma T cell responses (39, 40). Interestingly, Coughlin and coworkers describe a high frequency of circulating, functional survivin-specific CTLs, whereas intratumoral T cells were strikingly rare. Thus, survivin-based immunotherapy should indeed focus on T cell trafficking into tumor nests (30).

Immunologic clinical advances. Taken together, these preclinical studies have justified clinical testing of survivin for anticancer vaccination. Consequently, Survivin-directed immunotherapy has quickly been moved to the clinic. Recently, the first therapy-induced T cell responses against survivin were described. In a compassionate use setting, heavily pretreated patients with stage IV melanoma were vaccinated with an HLA-A2–restricted survivin epitope presented by autologous dendritic cells (41). These patients mounted strong T cell responses to this epitope as measured by ELISPOT assay. Furthermore, in situ peptide/HLA-A2 multimer staining revealed the infiltration of survivin-reactive CD8+ T cells into the soft tissue metastases of vaccinated patients. Notably, no vaccination-associated toxicity was observed. Hence, it is feasible to induce T cell responses against survivin; even in late stage tumor patients, and these vaccinations are well tolerated. In that regard, Pisarev et al. recently showed that survivin-directed CTL do not affect hematopoietic colony formation of CD34+-purified progenitor cells (42). In addition, in a patient with melanoma in complete remission following interleukin 2–based immunotherapy, a longitudinal examination of anti-survivin reactivity was made exceeding 7 years. Survivin-specific T-cell reactivity was found at all time points examined over the 7-year period. The data showed that anti-survivin T cells may persist in the periphery for extended periods in the absence of clinical manifestation of disease as well as autoimmunity (43). Finally, when used in an oral DNA vaccine, the survivin-directed immune response affected both tumor cells and tumor-associated angiogenesis, eradicating pulmonary metastases without toxicity, including wound healing or fertility in preclinical studies (44).

The first example of a successful application of survivin-based vaccination in the clinical setting was recently described in a patient with pancreatic cancer (45). This patient had a complete remission of liver metastasis under vaccination with a HLA-A2–restricted survivin peptide together with an adjuvant. Data from several ongoing phase I/II trials targeting survivin for patients with advanced cancer will provide further information towards this notion. In this regard, several survivin-based vaccination trials are currently ongoing at different institutes, e.g., Istituto Nazionale Tumori (Milan, Italy), Würzburg University Hospital (Würzburg, Germany), and Herlev University Hospital (Herlev, Denmark).