The MHC Class I Molecule and Tumor Associated Stress
Tumor cells experience a number of cellular stresses that translate into genetic mutations and altered gene expression and protein processing. Mutations in proteins involved in cell signaling, tumor suppression and DNA repair can lead to constitutive signaling, abnormal cell proliferation, and accumulation of numerous mutations resulting in genome wide instability. Genetic instability is detrimental to any cell but particular levels of genetic instability seem to impart a selective advantage to tumor cells (Cahill et al., 1999). In addition tumors often experience nutrient deprivation, hypoxia and oxidative stresses as they grow larger. To counteract these stresses, cancerous cells upregulate the production of proteins that induce angiogenesis, such as Her2/neu (Konecny et al., 2004), protect the cells from reactive oxygen species and inhibit apoptosis, such as the inducible heme oxygenase-1 (Mayerhofer et al., 2004).
Additionally, tumors display classical or universal stress responses that are easily identifiable with other disease states, such as viral infection. Indeed, a number of cancers are induced by viral infection by such agents as Epstein Barr Virus and Human Papilloma Viruses. Universal stress induced responses identified in tumor cells include up-regulation of MHC class I, Hsp72, and the NK cell ligands MHC-like MICA and MICB. Interestingly, these common stress induced genes are located within the MHC class I loci of chromosome 6 (Collins, 2004). In addition, many transcriptional regulators and translational regulators are over-expressed by both tumors and virally infected cells, including Stat-1, p68 and eIF4G (Abbas et al., 2000; Bates et al., 2005; Clemens, 2004).
Tumor cells can exhibit a number of stress induced changes in their genetic code, gene expression, and processing of proteins. These changes within a tumor cell can create novel and unrealized MHC class I ligands to CTL that survey surrounding tissue for "unhealthy" cells. Stress-induced class I ligands have been associated with a variety of tumors and their role in the immune response against tumors is being actively explored. In addition, many stress related changes in class I presentation of self peptides observed in tumors cells are mirrored by virus infected cells. We will therefore focus our discussion of tumor induced changes in MHC class I presentation on characterizing (1) tumor-derived class I peptides associated with mutation and alterations in gene expression and (2) universal stress induced class I ligands found in both cancer and viral infections.
Unique tumor antigens, or neo-antigens, are presented when point mutations occur in gene coding regions of ubiquitously expressed proteins, which may or may not be directly involved in cellular transformation. These mutations may alter proteasomal processing, resulting in differential cleavage of novel and unique peptides bound for MHC class I presentation. Alternatively, these mutations can alter the primary amino acid sequence of an established class I peptide, creating a new epitope for CTL recognition. These unique antigens are tumor-specific and, although they play a significant role in the individual host's anti-tumor response (Lennerz et al., 2005), as of yet, they are not into immunotherapeutic targets. Nonetheless, common cell cycle neo-antigens (Lennerz et al., 2005), heat shock proteins (Gaudin et al., 1999), and apoptosis regulators (Mandruzzato et al., 1997) continue to be explored as stress-related, class I presented, immunotherapeutic targets. As an example of a mutation induced tumor change we will focus on caspase 8, a component of Fas and TNF mediated apoptosis pathway.
Caspase 8 is a cysteine protease required for the initiation of Fas and TNF mediated apoptosis. Frame-shift deletion mutations, resulting in loss of pro-apoptotic function, are common in hepatocellular carcinomas (Soung et al., 2005). An HLA-B*3503 restricted CTL epitope derived from a loss of function caspase 8 mutant was identified from a squamous cell carcinoma of the head and neck by the generation of a tumor specific CTL clone stimulated by the autologous tumor. The mutation affected the stop codon, resulting in the extension of the protein by 88 amino acids (Mandruzzato et al., 1997). The B*3503 CTL epitope includes five amino acids of the extension site. In this manner, the MHC class I can act as a reporter of mutations in critical, transforming elements in the development of tumors.
It is important to note the possibility of changes in post-translational modifications, such as glycosylation and phosphorylation, altering the class I peptide repertoire of cancerous cells. Mutations in the molecular chaperone Cosmc affects the proper folding and activity of the core 1 beta3-galactosyltransferase which results in alterations in normal O-glycosylation (Ju and Cummings, 2002). For example, the Muc-1 antigen is an under-glycosylated mucin expressed by a number of tumor types, and mouse CTL have been generated against this glycopeptide (Xu et al., 2004). In addition, the constitutive activation of receptor tyrosine kinases and cyclin dependent kinases result in increased phosphorylation of cellular targets. Mouse CTL have been generated against phosphorylated class I restricted peptides (Andersen et al., 1999). CTL have the ability to recognize phosphory-lated and glycosylated variants of peptide epitopes. Class I molecules are able to indirectly reflect tumor stress through modifications in proteins and their constituent peptides.
Gene Expression Induced Stress Related Changes: Reactivation of Repressed Genes and Over-expression
Alterations in gene expression can induce changes in peptide presentation, including the expression of germ cell genes, often referred to as cancer-testis (CT) genes. Normally, these genes are expressed in the trophoblast and in testicular germ cells, which do not express MHC class I (Van Der Bruggen et al., 2002). Peptides novel to the mature immune system are presented on the MHC class I upon processing of the protein products of these genes. The function of most CT gene products is unknown but they are putatively involved in spermatogenesis. Their activation is likely due to genome wide demethylation seen in many tumors (Loriot et al., 2006). This extensive demethylation may be due to the loss or downregulation of DNA methyltransferases, as a result of transcriptional disregulation or mutation. These events open up the promoter regions of numerous genes that are normally transcriptionally repressed. The MAGE, BAGE, and GAGE families of CT genes were first identified in melanoma but have since been recognized in numerous tumor types (Van Der Bruggen et al., 2002). Such antigens represent ideal targets for immunotherapy, as they are common to many tumor types and are tumor specific. The up-regulation of host genes during stress can also result in changes in MHC class I presentation. A low level of class I presentation of a particular peptide may be seen ubiquitously or may be tissue specific. Over-expression of these proteins may result in significant changes in the density of these peptides presented on the cell surface, which may facilitate CTL recognition. In cancer, commonly over-expressed proteins include cell cycle regulators, such as cyclin D1 (Dengjel et al., 2004), receptor tyrosine kinases such as Her-2/neu (Fisk et al., 1995), and apoptosis regulators, such as p53 (Ropke et al., 1996). It has been difficult to discern which of these over-expressed proteins are presented in greater density, if at all, on the cell surface. Therefore, it is difficult to predict whether these epitopes are sufficiently or overly immunogenic for immunotherapeutic targeting. For example, human telomerase reverse transcriptase (hTERT) is over-expressed in a number of tumors, yet, fails to produce an anti-tumor response in hTERT peptide vaccinated patients. Although hTERT is over-expressed, it has now been determined that the telomerase derived peptides are not presented on the MHC class I molecule of telomerase expressing tumors (Parkhurst et al., 2004). Thus, while it is likely that class I present stress related over-expressed peptides, such presentation must be carefully validated. As examples of gene expression induced changes in MHC class I presentation, we will focus on peptides derived from MAGE-A1, a germ cell gene reactivated in many melanoma tumors, and Heme Oxygenase-1, a stress induced enzyme up-regulated in several tumor types.
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