Autoantibodies as reporters identifying aberrant cellular mechanisms in tumorigenesis (original) (raw)
HCC is unusual in that it is possible to identify individuals, such as those with liver cirrhosis and chronic hepatitis, who are at high risk of developing the disease over a period of several years (26). During conversion from chronic liver disease to HCC, the percentage of autoantibody-positive individuals rises significantly. In one study (23), one-third of HCC patients for whom serial serum samples were available before and at the time of diagnosis showed changes in autoantibody status (Table 1). The changes took place in the background of an already elevated frequency of autoantibodies in patients with chronic liver disease, suggesting that additional immunogenic stimuli were associated with tumorigenesis. Most patients manifested neoantibody responses, while the others showed increases in titers of pre-existing autoantibodies.
Changes in autoantibody responses associated with transformation to HCC in chronic liver disease patients
Sera showing the presence of neoantibodies have been used to immunoscreen cDNA expression libraries in order to isolate the putative tumor-associated antigens, a technique that was earlier used to isolate autoimmune rheumatic disease antigens. Proteins isolated with such tumor-associated antibodies include a serine-arginine repeat-bearing protein that acts in alternative mRNA splicing (27) and a cell cycle–related protein that represents a subunit of protein phosphatase 2A (28–30). Although this technique has been abundantly successful in isolating and characterizing many tumor-associated antigens, including those associated with PND syndromes (5, 6) and cancer/testis antigens (31), it remains possible that some of these molecules were antigenic even before the development of the tumor. This distinction can be made directly when premalignancy sera are available for a given patient, although tumor specificity of the antigens can also be established by showing that antibodies are present predominantly in patients with cancer.
In early studies on HCC, the tumor-associated autoantigens identified appeared to be idiosyncratic features of individual patients (27, 28), but more recent work has identified antigen-antibody systems that occur at relatively high frequency among cancer patients. Thus, antibodies to the nuclear protein cyclin B1, which is expressed in the S and G2 phases of the cell cycle, have been found in 15% (15/100) of patients with HCC, but in the same cohort no antibodies were detected to cyclins D1 and E and only one patient had antibodies to cyclin A (32). This apparent dominance of autoimmunity to cyclin B1 among many cell cycle–regulating proteins is as yet unexplained, but the finding of interest here is the presence of autoantibody to a single self protein in a significant subset of patients.
Analysis of sera from a large cohort of Chinese HCC patients identified a common 62-kDa autoantigen, p62, which has since been cloned and identified. This protein carries two types of RNA-binding motifs, an RNP consensus sequence motif in the amino-terminal region and four repeats of an hnRNP-K homology (KH) motif in the C-terminal two-thirds of the protein (33). Twenty-one percent of this group of HCC patients had antibodies to p62, whereas patients with liver cirrhosis, patients with chronic hepatitis, and normal controls were negative, indicating that the autoimmune response was highly HCC-related. Several recent studies on related proteins have now added to the potential significance of these observations. Nielsen et al. (34), studying cellular proteins that bind to IGF-II mRNAs in a rhabdomyosarcoma cell line, isolated three proteins that bind the 5′ untranslated region found in one IGF-II mRNA species (leader 3 IGF-II mRNA), which is developmentally regulated and expressed in fetal tissue but not adult tissue. These IGF-II mRNA binding proteins, called IMP-1, -2, and -3, all contained the RNP consensus sequence and KH RNA-binding motifs. p62 and IMP-2 are alternatively spliced products of the same gene.
The relationship of this family of mRNA-binding proteins to cancer has been shown in two studies besides our own (Table 2). Mueller-Pillasch et al. (35) isolated a cDNA overexpressed in human pancreatic cancer by differential screening of cancer versus normal tissues. The deduced protein called Koc (KH protein overexpressed in cancer), which is identical to IMP-3, is overexpressed in several other types of cancer. Doyle et al., who had earlier isolated c-myc mRNA coding region instability determinant binding protein (CRD-BP), a murine homolog of human IMP-1 (Table 2) that binds a 250-nucleotide sequence in the coding region of c-myc mRNA, have recently shown amplification of the gene encoding this protein in human breast cancer (36). Previously, these investigators had shown in cell-free mRNA decay experiments that CRD-BP acts as a shield to protect c-myc mRNA from rapid degradation by binding to the 250-nucleotide instability determinant (37).
Messenger RNA-binding proteins with RNP consensus sequence (RNA recognition motifs) and KH (hnRNP-K homology) domains involved in tumorigenesis
The studies showing that p62 and Koc are mRNA-binding proteins and that one of the targets is IGF-II mRNA have interesting implications with regard to cancer. IGF-II is a growth factor whose overproduction has been shown to promote tumorigenesis (38), and its mRNA is specifically overexpressed in HCC tumor nodules (39). IGF-II transgenic mice have increased frequency of diverse malignancies (40), and in SV40 oncogene-induced tumorigenesis, IGF-II serves as an accessory factor in transformation (41). Similarly, as Pasquinelli et al. (42) have observed, transgenic mice expressing the hepatitis B virus envelope exhibit chronic hepatocellular injury and inflammation, leading to successive cycles of regenerative hyperplasia and inflammation and ultimately to development of HCC. These authors found no abnormalities in the structure or expression of a large panel of oncogenes and tumor suppressors, including ras, myc, fos, abl, src, Rb, and p53, but noted that IGF-II was consistently overexpressed.
These data suggest that IGF-II induction is common to HCC tumors induced by a variety of stimuli and raise the question of whether the IGF-II mRNA dysregulation is a necessary step in this disease pathway. Indeed, we have observed by immunohistochemistry that in one-third (9/27) of patients with HCC, cancer nodules showed abundant immunostaining for p62 protein whereas adjacent nonmalignant hepatic parenchymal and other cells were devoid of detectable p62 (Figure 1a). In one of two cases of cholangiocarcinoma, malignant bile duct epithelial cells also expressed p62 (Figure 1b). Nine specimens of normal human adult liver did not contain detectable p62, whereas fetal liver contained immunoreactive p62 in hepatic parenchymal cells (43). Hence, p62 is a developmentally regulated cellular protein expressed in fetal liver but not in adult liver and aberrantly expressed in HCC. We propose that, analogous to the manner in which CRD-BP protects c-myc mRNA from degradation, p62 and Koc might allow expression of fetal IGF-II mRNA in malignant cells, resulting in overproduction of IGF-II growth factor.
Tumor nodule from a patient with HCC examined by immunohistochemistry (a), showing expression of p62 protein in cytoplasm of cancer cells but no detectable p62 in hepatic or other cells in adjacent noncancer areas. One of two cholangiocarcinoma patients showed expression of p62 in cytoplasm of malignant bile duct cells (b).