Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer

Genomic imprinting is an epigenetic modification that leads to the preferential or exclusive expression of a gene from one of the two parental alleles in somatic cells [1]. Abnormal imprinting involved in a number of human diseases, particularly, LOI is one of the most frequent genetic alterations in cancers [2]. LOI can result in either activation or silencing of the normally silent or expressed allele of a growth promoting gene or a growth inhibitory gene, respectively. Research suggests that disruption of imprinting mechanisms may play a critical role in the development of cancer [3]. The cluster of imprinted genes on human chromosome 11p15.5 comprises two imprinted domains: the IGF2-H19 domain and the KCNQ1 domain [4]. H19 and IGF2 genes are imprinted genes and expressed differently depending on whether they are carried by a chromosome of maternal or paternal origin [5]; normally IGF2 expression is coordinately regulated with the maternally expressed H19 gene that produces a noncoding RNA. But in bladder cancer, paternal hypomethylation leads to biallelic H19 expression [6], whereas in Wilms'tumor, maternal hypermethylation and biallelic IGF2 expression are common [7, 8]. The level of H19 RNAs in Wilms'tumor is also found to inversely correlate with levels of IGF2 mRNA [9], H19 RNAs were found in polysomes, indicative of H19 translation and/or potential transregulation of IGF2 translation. The upstream promoter region of H19 has the imprinting-control region (ICR) or CTCF binding sites, where the methylation status of this region is critical to the regulation of imprinting of the H19/IGF2 locus located in chromosome 11p15 [10]. LOI of IGF2 is coupled to abnormal H19 methylation in the Wilms tumor case [11]. There may also be an independent mechanism for regulating IGF2 in Beckwith-Wiedemann syndrome (BWS) patients [12]. IGF2 encodes a potent mitogenic growth factor that is active in early development and plays an important role in embryonic and fetal growth [13]. Increased expression of IGF2 is a common feature of both pediatric and adult malignancies since IGF2 binds to the IGF1 receptor to initiate intracellular signaling cascades that lead to cell proliferation [14]. IGF2 stimulates cell proliferation and development in normal human growth. Study showed the overexpressed IGF2 gene is a growth factor for tumors mediated through both the paracrine and autocrine pathways in human cancers. The IGF2 gene may thus play an important role in lymph vessel permeation especially in expanding-type gastric cancers [15]. LOI of IGF2 gene is an important cause of biallelic expression of IGF2 and has been reported in many different types of tumors including osteosarcoma [16], lung adenocarcinomas [17], head and neck squamous cell adenocarcinomas [18], Wilms'tumor [7], prostate cancer [19], and colorectal carcinomas [20]. Studying mice with Apc-Min/+ model of human familial adenomatouspolyposis showed excessive expression of IGF2 resulted increase in the number and the diameter of colon adenoma and increased susceptibility to colon carcinoma [21]. Moreover LOI of IGF2 might provide a marker for identifying an important subset of the population with cancer or at risk of developing cancer [22]. Normally the KvDMR1 in intron 10 of KCNQ1 unmethylated paternally promote LIT1/KCNQ1OT1 expressed paternally antisense RNA [23]. The human LIT1 transcription unit lies within the 11p15.5 imprinted gene cluster and functions as non-coding RNA [24]. Aberrations of LIT1 expression, such as those caused by LOI, involving aberrant hypomethylation and activation of the normally silent maternal allele and LOI IGF2 have been observed in Beckwith-Wiedemann syndrome (BWS) and colorectal cancer [23, 25]. In addition, loss of maternal-specific methylation at the LIT1 locus in BWS and several cancers correlates with abnormal imprinting status of CDKN1C [26]. Soejima et al. have recently shown that loss of CpG and histone H3 methylation at a differentially methylated region (DMR)-LIT1 leads to a reduction of CDKN1C expression in esophageal cancer [27]. LOI of IGF2 in gastric tumour tissue except from Taiwan in Chinese and in Japanese patients [15, 28] and the clinicopathological features of gastric cancers with LOI of has been reported rarely. In this study LIT1, IGF2 and H19 imprinting status in gastric cancer (GC) which is the second most common cause of cancer-related death in the world was investigated and whether LOI of LIT1, IGF2 and H19 are associated with clinicopathological features was evaluated. The study showed LOI of IGF2 is associated with gastric corpus and LNM in gastric cancer tissues, suggesting that IGF2 plays an important role in gastric carcinogenesis.

The cluster of imprinted genes on human chromosome 11p15.5 consists of two domains: IGF2-H19 domain and the KCNQ1 domain [4]. LOI of IGF2 has been observed in 10% of the lymphocytes from normal individuals [30]. In normal human brain, biallelic expression of IGF2 and/or H19 is found despite differential methylation and CTCF binding [31]. In this study, we have shown that LOI of the LIT1, IGF2 and H19 are present in 54.6%, 45% and 8.6% of gastric cancer tissues in Chinese patients respectively. This is the first study to detect on the LOI of LIT1, IGF2 and H19 in gastric cancer in China-Mainland patients and LOI of IGF2 positive correlation with gastric corpus tumour (OR = 8, 95%CI = 1.425-44.920, p = 0.018) and lymph node metastasis (OR = 4.5, 95%CI = 1.084-18.689, p = 0.038).

The frequency of IGF2 LOI (+) gastric cancers (45%, 18/40) is slightly higher than that reported from Taiwan (34.5%, 10/29) [28]. High frequency of IGF2 LOI was observed in tumor and adjacent normal tissues and Igf2 LOI with Apc+/Min mice showed a shift toward less differentiation and an increase in tumor initiation indicating that IGF2 LOI occur at an early stage in cancer development [32]. Although the mechanisms underlying IGF2 LOI in human cancer remains unknown, it is likely to directly or indirectly involve the H19 ICR. We used the allele-specific restriction enzyme digestion technique to identify LOI status, similar to that reported previously [22]. Some papers have explained the mechanisms in the tumourigenesis of gastric carcinogenesis [33]. A variety of epigenetic alterations in human cancers include global DNA hypomethylation, gene hypomethylation and promoter hypermethylation, and IGF2 LOI. The mechanisms for LOI are hypermethylation or hypomethylation of a DMR upstream of the H19 gene, allowing activation of the normally silent maternal allele of IGF2. LOI may precede the development of cancer and may thus serve as a common marker for risk, but also as a model for understanding the developmental mechanism for normal imprinting. Therefore, it is possible that IGF2 LOI play a role in the tumourigenesis through epigenetic modification of DMR. Positive correlations were identified between elevated IGF2 expression and hypermethylation of CTCF binding sites at the H19 proximal imprint center in ovarian cancer [34]. H19 may be a tumor suppressor gene involved in head and neck carcinogenesis [35]. Epigenetic alterations of H19 or LIT1, which encode untranslated RNAs on 11p15, are strongly associated with cancer risk or specific birth defects in BWS [36].

We found that gastric corpus cancer is associated with higher IGF2 positive LOI rate, while Liou et al [37] found that proximal colon cancer is independently associated with higher positive LOI rate, consistent with a recent report from Japan [38]. However, larger population are needed to screen whether IGF2 LOI is involved in which pathways of gastric carcinogenesis. LOI of LIT1 involves aberrant hypomethylation and activation of the normally silent maternal allele. Our data suggest that LIT1 LOI may be associated with gastric cancer tumorigenesis. Histone modifications and DNA methylation are important for the regulation of LIT1 expression to form active or repressive chromatin structure [27]. LIT1 is a non-coding RNA, like Xist, Tsix and Air, LIT1 RNA plays a critical role in the bidirectional spreading of inactive chromatin structures [39], silencing imprinting genes [40] and formating of the imprinting center (IC) to coordinate imprinting in the 11p15.5 region. Timing of LIT1 RNA expression is vital for the proper initiation of imprinting genes [41, 42]. Premature termination of the LIT1 transcript leads to LOI in the proximal region indicating full-length Lit1 RNA is necessary for maintaining the imprinting status [43]. Mouse Lit1 RNA plays a critical role in silencing at the IC of the imprinted gene cluster and the transcript length of Lit1 RNA is important for the degree of silencing [44].

And we found patients with LOI of IGF2 in their tumour had higher increased risk of the lymph node metastasis than those without (OR = 4.5, 95%CI = 1.084-18.689, p = 0.038). Recently our study found metastatic lymph node ratio is a useful independent prognostic factor and may obviate possible confounding factors that are related to stage migration, and should be considered as an important component in the lymph node ategory. Lymph node ratio category has advantages in providing a more precise prognostic value than the pN category (5th edition, UICC). We recommend that classification of nodal status be established by a combination of both the metastatic nodes number and ratio, which would be the best category to provide both rational lymph node dissection and the foundation for adjunctive therapy and predict the prognosis [45]. Ohashi et al reported conventional pathological factors, such as tumor size, depth of submucosal invasion, and lymphatic invasion, have a significant influence on lymph node metastasis in submucosal invasive gastric cancer [46]. Li et al showed depth of invasion, lymph node metastasis, hepatic and peritoneal metastasis and surgical curability were significant factors affecting survival of the gastric carcinoma patients [47]. But we failed to find such an association. Liu et al found transversal and skipping metastases of sentinel lymph nodes (SLN) are notable and therefore rational lymphadenectomy should be performed in primary gastric cancer [48]. Some research demonstrated lymph node metastasis were independent prognostic factors in human gastric carcinoma [49]. And high expression of mitotic centromere-associated kinesin (MCAK) and tripartite motif-containing 29 (TRIM29) are predictors for lymph node metastasis [50, 51]. It might be more appropriate that identifying patients at high risk of lymph node metastasis who should be offered gastrectomy rather than endoscopic mucosal resection, because patients with lymph node metastasis are more likely to express IGF2 LOI than those without. Our result was consistent with other studies that LOI of IGF2 is also important in the carcinogenesis [15, 28].

In all, high frequency of IGF2 LOI is present in patients with gastric cancer in the northeast of China. The association of IGF2 LOI with lymph node metastasis may contribute to the development and progression of gastric cancer.