J. Keith Killian

M6P/IGF2R Imprinting Evolution in Mammals

Imprinted gene identification in animals has been limited to eutherian mammals, suggesting a significant role for intrauterine fetal development in the evolution of imprinting. We report herein that M6P/IGF2R is not imprinted in monotremes and does not encode for a receptor that binds IGF2. In contrast, M6P/IGF2R is imprinted in a didelphid marsupial, the opossum, but it strikingly lacks the differentially methylated CpG island in intron 2 postulated to be involved in imprint control. Thus, invasive placentation and gestational fetal growth are not required for imprinted genes to evolve. Unless there was convergent evolution of M6P/ IGF2R imprinting and receptor IGF2 binding in marsupials and eutherians, our results also demonstrate that these two functions evolved in a mammalian clade exclusive of monotremes.

M6P/IGF2R is mutated in squamous cell carcinoma of the lung

In addition to the intracellular sorting of lysosomal enzymes, the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) plays a critical role in regulating the bioavailability of extracellular proteolytic enzymes and growth factors. It has also been shown to be mutated in a number of human cancers, and to suppress cancer cell growth. The purpose of this study was to determine if the M6P/IGF2R is mutated in lung cancer, a leading cause of cancer death worldwide. Archival pathology specimens were obtained on 22 patients with newly diagnosed, untreated squamous cell carcinoma of the lung. Two polymorphisms in the 3′-untranslated region of the M6P/IGF2R were used to screen lung tumors for loss of heterozygosity (LOH) by PCR amplification of DNA. Nineteen of 22 (86%) patients were informative (heterozygous), and 11/19 (58%) squamous cell carcinomas of the lung had LOH at the M6P/IGF2R locus. The remaining allele in 6/11 (55%) LOH patients contained mutations in either the mannose 6-phosphate or the IGF2 binding domain of the M6P/IGF2R. Thus, the M6P/IGF2R is mutated frequently in squamous cell carcinoma of the lung, providing further support for its function as a tumor suppressor.

Imprint status of M6P/IGF2R and IGF2 in chickens.

Abstract. Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.

M6P/IGF2R loss of heterozygosity in head and neck cancer associated with poor patient prognosis

BackgroundThe mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes for a multifunctional receptor involved in lysosomal enzyme trafficking, fetal organogenesis, cytotoxic T cell-induced apoptosis and tumor suppression. The purpose of this investigation was to determine if the M6P/IGF2R tumor suppressor gene is mutated in human head and neck cancer, and if allelic loss is associated with poor patient prognosis.MethodsM6P/IGF2R loss of heterozygosity in locally advanced squamous cell carcinoma of the head and neck was assessed with six different gene-specific nucleotide polymorphisms. The patients studied were enrolled in a phase 3 trial of twice daily radiotherapy with or without concurrent chemotherapy; median follow-up for surviving patients is 76 months.ResultsM6P/IGF2R was polymorphic in 64% (56/87) of patients, and 54% (30/56) of the tumors in these informative patients had loss of heterozygosity. M6P/IGF2R loss of heterozygosity was associated with a significantly reduced 5 year relapse-free survival (23% vs. 69%, p = 0.02), locoregional control (34% vs. 75%, p = 0.03) and cause specific survival (29% vs. 75%, p = 0.02) in the patients treated with radiotherapy alone. Concomitant chemotherapy resulted in a better outcome when compared to radiotherapy alone only in those patients whose tumors had M6P/IGF2R loss of heterozygosity.ConclusionsThis study provides the first evidence that M6P/IGF2R loss of heterozygosity predicts for poor therapeutic outcome in patients treated with radiotherapy alone. Our findings also indicate that head and neck cancer patients with M6P/IGF2R allelic loss benefit most from concurrent chemotherapy.

Genomic structure of the human M6P/IGF2 receptor

The human mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) gene located at 6q26 (Rao et al. 1994) encodes for a multifunctional receptor that possesses distinct binding sites for phosphomannosyl glycoproteins and IGF2 (MacDonald et al. 1988; Morgan et al. 1987), retinoic acid (Kang et al. 1997), and urokinase-type plasminogen activator receptor (uPAR; Nykjaer et al. 1998). The receptor is involved in fetal development, tumor suppression, maternal regulation of intrauterine growth, and has recently been associated with the development of human cognitive ability (Chorney et al. 1998; De Souza et al. 1997; Kornfeld 1992; Wang et al. 1994). To facilitate further genetic analyses of the human M6P/IGF2R, we have determined its complete genomic organization, defined the intron-exon boundary sequences, and designed intronic oligonucleotides for PCR amplification of the 48 exons included in the 136-kb genomic sequence. The M6P/IGF2R is a chimeric receptor possessing binding sites for four distinct classes of ligands (Kang et al. 1997; Nykjaer et al. 1998; MacDonald et al. 1988; Morgan et al. 1987). The M6P binding site enables the M6P/IGF2R to target to the lysosomes both newly synthesized lysosomal enzymes from the Golgi and phosphorylated proteolytic enzymes endocytosed from the extracellular environment (Kornfeld 1992). Furthermore, the latent complex of transforming growth factor beta (TGFb), a potent growth inhibitor, binds to the M6P/IGF2R through these M6P binding sites, thereby facilitating its activation by plasmin (Dennis and Rifkin 1991). The effectiveness of TGFb activation may be further enhanced by the direct binding of uPAR to the M6P/IGF2R (Nykjaer et al. 1998). The mammalian M6P/IGF2R also contains an independent IGF2 binding site that is absent in chickens (Zhou and Sly 1995). IGF2 binding to the M6P/IGF2R leads to lysosomal degradation rather than intracellular signaling, a process mediated by both the IGF1 and the insulin receptors (Kornfeld 1992). Therefore, loss of M6P/IGF2R function would be predicted to increase the extracellular concentration of IGF2, decrease the level of active TGFb, and increase the secretion of proteolytic enzymes. These biological effects suggest the M6P/IGF2R functions normally as a tumor suppressor. A number of reports now strongly support this postulate for a wide variety of human tumors. Briefly, the M6P/IGF2R is mutated in 60% of dysplastic liver lesions and HCCs in patients with or without hepatitis virus (HV) infection (De Souza et al. 1995a, 1995b; Yamada et al. 1997). Its inactivation also plays a prominent role in the early stage of breast cancer development (Hankins et al. 1996), and increased expression of the wild-type receptor in breast cancer cell lines leads to apoptosis (Oates et al. 1998). Furthermore, the M6P/IGF2R gene contains a poly-G region that is a common mutational target in colon, gastric, and endometrial tumors with mismatch repair deficiences and microsatellite instability (Souza et al. 1996). Thus, M6P/IGF2R inactivation is a frequent oncogenic event that occurs early in carcinogenesis. A full parental complement of autosomal genes is inherited by all offspring, but not all are biallelically expressed. The phenomenon of monoallelic expression of the same parental allele is called genomic imprinting (Surani 1998). The M6p/Igf2r gene is imprinted in mice (Barlow et al. 1991) and is expressed only from the maternal allele in all tissues except potentially the brain (Hu et al. 1998). In contrast, imprinting of the M6P/IGF2R gene is a polymorphic trait in humans, with most people having biallelic expression in all tissues (Xu et al. 1993). Although imprinting of the M6P/IGF2R may have provided an evolutionary advantage to the mother during the emergence of mammalian intrauterine growth (Haig and Graham 1991), monoallelic expression of this gene would also be predicted to increase cancer susceptibility. In support of this postulate, Xu et al. and associates (1997) have recently demonstrated M6P/IGF2R imprinting in 50% of Wilm’s tumors. Plomin and his colleagues have also identified the M6P/IGF2R as a putative ‘‘IQ gene’’ (Chorney et al. 1998). By comparing Caucasian children with an IQ of 160 or higher with those with an average IQ, they showed that the M6P/IGF2R is linked to human cognitive ability. If a polymorphism in the M6P/IGF2R coding sequence or its regulatory region is subsequently shown to functionally affect IQ, it would be the first identified gene known to contribute to intelligence. Thus, the M6P/IGF2R plays a fundamental role in biological processes ranging from embryogenesis to carcinogenesis. How the M6P/IGF2R could possess such diverse biological functions is an intriguing enigma that remains to be solved. To facilitate future studies on the human M6P/IGF2R, we have characterized its complete genomic structure. The human cDNA sequence was divided into 48 exons based upon the mouse genomic structure (Szebenyi and Rotwein 1994), and PCR primers were designed to traverse these putative introns. All the introns except 1, 2, and 4 were amplified from human genomic DNA with either long-template PCR (Boehringer Mannheim Corp., Indianapolis, Ind.) or traditional PCR (Qiagen, Inc, Santa Clarita, Calif.). PCR products were sequenced on an ABI automated sequencer (Perkin-Elmer Corp., Foster City, Calif.). The sizes of introns 1 and 2 and their 58 and 38 boundary sequences were previously defined (Riesewijk et al. 1996; Smrzka et al. 1995); this information is available on GenBank (Accession Nos. X83699, X83700, and X83701). To sequence intron 4, human BAC clones (174E20 and 650K6) were first obtained by probing filters from Research Genetics, Inc. (Huntsville, Ala.) with an RT-PCR product spanning exons 1 to 6. A 13-kb stretch of DNA containing intron 4 was then PCR amplified from this BAC clone and the exon-intron boundaries sequenced. With the exception of intron 15, all exon-intron splice sites (93/94) conformed to the AG/GT rule (Mount 1982). Interestingly, the single intron splice site exception is also present in the mouse M6p/Igf2r homolog (Szebenyi and Rotwein 1994). Intron sizes were determined by sequencing the entire intron or estimated by electrophoresis. Twenty introns were sequenced entirely (that is, introns 8, 9, 13–17, 21, 23, 24, 27, 28, 32–34, 37, 38, 42, 43, and 46), and at least 100 bp of flanking intronic sequence were determined for the remaining introns. The entire human M6P/ IGF2R gene was then reconstructed with the use of the previously Correspondence to: R.L. Jirtle Mammalian Genome 10, 74–77 (1999).

Polymorphisms, genomic imprinting and cancer susceptibility.

Polymorphisms have been identified in proto-oncogenes and tumor suppressor genes that predispose people to cancer. Recent evidence indicates that genomic imprinting, an epigenetic form of gene regulation that results in uniparental gene expression, can also function as a cancer predisposing event. Thus, cancer susceptibility is increased by both Mendelian inherited genetic and non-Mendelian inherited epigenetic events. Consequently, chemical and physical agents cannot only induce cancer through the formation of genetic mutations but also through epigenetic changes that result in the inappropriate expression of imprinted proto-oncogenes and tumor suppressor genes. The role of genomic imprinting in carcinogenesis and cancer susceptibility is examined in this review.