Background

Breast cancer ranks first among female cancer incidence. In recent years, the incidence of breast cancer in China has been rising rapidly, exceeding the world average growth level (2%), and the growth rate of big cities such as Beijing and Shanghai has even reached about 5%. Breast cancer has become the most common malignant tumor in women in China, with 269,000 new cases of breast cancer and 70,000 deaths each year. Insulin-like growth factor 2 (IGF2) was cloned from an adult DNA library by Bell et al. (1984). IGF2 is a protein hormone that regulates cell proliferation, growth, migration, differentiation and survival (Bergman et al., 2013; Coto et al., 2018). IGF2 is preferentially expressed in many somatic cells during early embryonic and embryonic development. At present, IGF2 can be detected in circulating plasma, and its detection rate in fetal circulating blood is the highest. IGF2 is also an imprinted gene, expressing a paternal allele. But it is a double allele expression in adult liver and central nervous system (Tahara et al., 2017). This article reviews the structure, mechanism, imprinting status of IGF2 gene and its relationship with breast cancer.

1 IGF2 Gene Family, Structure and Function

1.1 IGF2 gene structure

IGF2 gene is a polypeptide composed of 67 amino acids (Figure 1), produced by hepatocytes and closely related to insulin. IGF2 gene is located on chromosome 11p15.5 next to insulin gene, spanning 30 KB (Ben-Zaken et al., 2017). IGF2 gene initially synthesized IGF2 protein precursor, which was composed of A-E domain and a 24-residue signal peptide. Signal fetal cleavage is a large number of IGF2 precursors. After translation, O-glycosylation of residue E domain is carried out, which can promote the further processing of precursors. The precursor of IGF2 loses E domain through continuous protein hydrolysis and becomes mature IGF2. Incomplete treatment of IGF2 precursors leads to the inclusion of all or part of the E domain in the polypeptide, collectively known as the large IGF2 gene, which secretes into the blood, accounting for 10-20% of the normal IGF2 gene. Glycosylation of large IGF2 gene promotes the formation of ternary complexes in serum. Large IGF2 gene can also promote the formation of binary complexes IGFBP2, IGFBP5 and IGFBP3. IGF2 gene transcription depends on the four promoters of P1-4. During embryonic development, the expression and transcription of single allele comes from P2-P4. In adult liver, there is expression of P1 promoter (Callum, 2013). In the process of growth and development, the biological activities of the four promoters are different, tissue-specific and related to developmental stages.

1.2 Mechanisms of IGF2

IGF2 cannot exist alone in blood after secretion. It must be bound to target cells by IGFBPs and bind to insulin-like growth factor receptor (IGFR) on the surface of target cells. Only through PIK3/AKt and Ras/MAPK 2 signaling pathways (Figure 2) can it play its biological role (Massoner et al., 2010; Unger et al., 2017). There are two types of insulin-like growth factor receptors, type I and type II. Type I receptor is a heterosexual transmembrane receptor. It consists of two alpha subunits of extracellular matrix and two beta subunits of intracellular matrix connected by disulfide bond. Type I receptors bind to ligands, triggering tyrosine-specific phosphorylation of receptors and subsequently producing various physiological reactions. Type II receptor is a single chain protein located on almost all cell surfaces, which is related to lysosomal enzymes and classification of cell phagocytosis.

1.3 IGF2 gene imprinting

Gene imprinting does not conform to Mendel’s law of inheritance. Loss of gene imprinting (LOI) is a common phenomenon in tumors. The activation of maternal alleles silenced by IGF2 under normal conditions is a typical example in cancer. IGF2 encodes an important autocrine growth factor (Mishima et al., 2015). Mouse IGF2 gene is the first endogenous imprinted gene identified. It is expressed only in paternal alleles and regulated by enhancers, DNA differential methylation domains (DMD) and promoters (Zhao et al., 2016). Usually, only the paternal IGF2 gene is expressed and the maternal IGF2 gene is turned off, but the abnormal expression of the maternal IGF2 gene can cause the abnormal binding of insulator CTCF to DMD, which is caused by the methylation of damaged CTCF or DMD. IGF2 imprinting loss (IGF2 LOI) as a marker has been widely studied in various human tumors. It has been reported that abnormal imprinting of IGF2 is closely related to childhood tumors such as Wilms’ tumors and some adult tumors such as renal clear cell sarcoma and ovarian cancer (Tserga et al., 2017). LOI leads to overexpression of IGF2 gene, which activates IGF1R and AKT1, which is a powerful driving force for cell proliferation (Belharazem et al., 2016). Venkatraman et al. (2013) demonstrated that the up-regulation of H19-IGF2 gene locus plays a role in the regulation of long-term hematopoietic stem cell function, the down-regulation of hematopoietic stem cell activity and cell proliferation (Venkatraman et al., 2013).

2 Cloning and Expression of IGF2 Gene

Using the identical sequences of IGF1 and IGF2 as probes, Bell et al. cloned IGF2 from human liver DNA Library in 1984 and deduced a 180-amino-acid IGF2 protein precursor, which contains N-terminal signal sequence followed by mature peptide and C-terminal extension, i.e. E region. Both signal sequence and D region were hydrolyzed by proteins and then migrated to form mature 67 amino acid peptide. A Buffalo rat liver IGF2 gene was used as a probe. Dull et al. cloned human IGF2 from human embryonic liver gene library in the same year. It is inferred that the precursor of IGF2 of 180 amino acids starts at the middle 24 and its molecular weight is 20.1 kD (Williams et al., 2013). Using the coding region of human liver IGF2 as a probe, Shen et al. cloned IGF2 from human embryonic DNA Library in 1988. They identified four embryonic variants, different from liver variants, with only 5’end primers and nearly identical coding sequences. RNA imprinting analysis revealed transcripts 6.0, 4.9 and 3.2 kb in embryos using special exon probes. The 4.9 KB variant at the 5-terminal is different from the liver transcript and other embryonic transcripts.

De Pagter-Holthuizen et al. (1987) elucidated that 5.3-kb mRNA originated in exon 1 of IGF2 and expressed in adult liver, while 6.0-kb transcript originated in exon 4 and expressed in embryonic tissues and some adult non-liver tissues. They concluded that the 2.2-kb transcript also started at exon 4. 4.8-kb transcript originates from exon 4B, which is expressed in most embryonic tissues and some adult non-liver tissues. RNA imprinting analysis using a 3’-terminal primer UTR probe revealed an abnormal 1.8-kb transcript in the liver of adults and embryos, adrenal glands, skeletal muscles and kidneys of embryos, but not in the lungs and brain tissues of embryos. Sequence analysis showed that the transcription originated from exon 7 of IGF2 gene. In vitro translation results showed that it was an 84-amino acid peptide with an apparent molecular weight of 8.3 kD.

Monk et al. identified five IGF2 transcripts, which differed only in the untranslated region of their 5’primers. RNA imprinting analysis revealed that the transcript was initiated by the P1 promoter and existed only in the liver, known as the P1 transcript. P0 transcripts are diverse in most tissues except the brain and early placenta. High expression exists in fetal skeletal muscle, full-term embryo and kidney. In contrast, the mouse P0 transcript was only expressed in the placenta. Monk et al. also identified two combined read-through transcripts containing INS2 gene exons from upstream mixed with IGF2 exons. Later in the study, it was generally named INSIGF.

3 Study on IGF2 in Breast Cancer

Breast cancer is a systemic disease, and its occurrence and development is a complex process involving multiple factors. Many bioactive substances are involved in the carcinogenesis and metastasis of breast cells. Abnormal signal transduction pathways mediated by these bioactive substances can cause excessive amplification of some genes, which can lead normal cells to accept abnormal proliferation, differentiation and growth signals, and ultimately promote the carcinogenesis of breast cells. IGF2 gene plays a role in the development of breast cancer through its unique signaling pathway (Li et al., 2018).

3.1 Stimulating effect of IGF2 on breast cancer cells

Margit Pacher’s experiment (Pacher et al., 2007) used an Afymetrix gene chip containing 45,000 probes to screen the whole genome of long-term IGF1/2 target genes. The up-regulation of IGF signal composition is closely related to amino acid transport and metabolism, protein biosynthesis and stability, nucleic acid base and glutathione synthesis of most genes. All these processes are also based on the absolute requirement of maintaining accelerated growth and value-added rates, which are exactly the survival markers of cancer cells. These combined metabolic effects, together with their strong mitotic activity, can make IGF signal abnormally activated into an extremely powerful oncogenic switch. It has been proved that overexpression of IGF1/2 alone can accelerate cell cycle progression and induce MCF7 breast cancer cell growth. It can also significantly promote cell growth during normal development.

3.2 IGF2 gene hypomethylation and breast cancer

IGF2 gene is mainly expressed in paternal lines. Previous studies have shown that LOI of IGF2 gene is observed in 30-70% of breast cancer patients. LOI of IGF2 promoter proximal sequence (DMRO) such as low methylation-related imprinting deletion (LOI) is considered as a susceptible biomarker of colorectal cancer. The Yoko Ito (Ito et al., 2008) study assessed whether DMRO methylation of IGF2 gene occurred before or after the onset of cancer by pyrophosphatic acid sequencing. The study analyzed DNA samples from 22 cases of breast cancer and 42 cases of colorectal cancer from cancer and non-cancer tissues, as well as peripheral blood samples from patients with colorectal cancer (n=192, control 96), breast cancer patients (n=364, control 96) and prospective European cancer survey [EPIC-96]. Norfolk (n=228 in breast cancer group, 225 in colorectal cancer group and 895 in control group). The results showed that the methylation level of IGF2 gene DMRO in tumors was lower than that in non-tumorous tissues. The detection rate of DMRO hypomethylation in breast (33%) and colorectal cancer (80%) tissues is higher than that in LOI. DMRO hypomethylation reaction of IGF2 gene is related to the risk of breast cancer, and can be used in the diagnosis of breast cancer.

Another Preetha J. experiment (Shetty et al., 2011; Creemers et al., 2016) was designed to discuss the role of methylation in the transcription and protein expression of IGF2. The methylation of IGF2 gene in the CpG region of allele-specific DNA can be called methylation differential regions (DMRs). This study revealed a significant deletion of methylation (P<0.000,1) in exon 9CpG cluster of IGF2 gene in 85% breast cancer tissues and adjacent 3% normal breast tissues. This study focused on the methylation of exon 9GpG cluster, which can be used as a biomarker to enhance the expression of IGF2 in breast cancer tissues in DMR2 region. The results suggest that IGF2 may also play a role in the transcription and expression of breast cancer.

3.3 Free IGF2 level and breast cancer

This study investigated the relationship between IGF2 and breast cancer. In the circulatory system of patients with limited and early breast cancer, the level of free IGF1/IGF2 increased significantly, the level of total IGF2 decreased, and the level of IGBPs changed. Despite the low incidence, it can be seen that IGF system plays a role in disease progression. IGF2 receptors have a high affinity with IGF2, which can eradicate other growth factors and reduce extracellular IGF2 levels. Studies on breast cancer tissues showed that the expression of IGF2 was increased. In the transition zone between normal epithelium and tumor tissues, stromal cells and leukocytes expressed IGF2, which might stimulate lymphocyte formation. Therefore, the level of IGF2 in extracellular environment was closely related to tumorigenicity. The addition of IGF2 in cancer tissues could stimulate cell proliferation, while the expression of IGF2 receptor transfected cancer cells. Inhibiting its growth and increasing apoptosis. Studies have shown that circulating free IGF2 levels are correlated with breast cancer size, but paradoxically, the level of circulating free IGF2 in cancer patients is lower than that in the control group. It is noteworthy that these findings become apparent only after correcting changes in body fluid composition after surgery. The serum level of IGFBP2 in cancer patients is lower than that in control group. This binding protein is an important determinant of free IGF2 level. Lower IGFBP2 may increase the free fraction of IGF2. This experiment concluded that free IGF2 may respond to the increase of biological activity of this axis in women with breast cancer, confirming that the production of IGF2 in breast cancer can promote local growth and increase the anti-apoptotic signal (Espelund et al., 2008).

3.4 IGF2 and breast cancer susceptibility in special population

The experiment compared that African-American (AA) women with breast cancer were more likely than Caucasian (CA) women to change for advanced diseases, with higher risk of recurrence and worse prognosis. The expression of IGF2 in paired breast tissue samples was higher in AA women than in CA women. IGF2 can induce strong mitogen of cell proliferation and survival signal by activating IGF1 and IGF1R (IGF1 receptor). The level of IGF2 cycle is regulated by cell uptake of IGF2R. IGF2R plays a role in maintaining the level of IGF2 in target tissue. Overexpression of IGF2 in MCF-7 breast cancer cells can increase the secretion of lysosomal cathepsin D. Its secretion and prognosis are poor and metastasis increase phase. It can also reduce the disease-free survival of breast cancer patients. The expression of IGF1R in normal AA tissues was significantly higher than that in normal CA tissues. The expression of IGF1R in normal AA tissues was similar to that in malignant AA tissues. It was concluded that the difference in the expression of IGF1R and IGF2R might increase the risk of malignant transformation of breast in young AA women and contribute to the more aggressive phenotype of breast cancer (Kalla et al., 2010; Dong et al., 2015).

The lifetime risk of breast cancer in BRCA1 and BRCA2 gene mutation carriers is 40%-80%, indicating that there is a risk modifier (Xu et al., 2016). Previous studies have shown that there is an important correlation between IGF signaling pathway and gene mutation. In this study, whether the additional IGF signaling gene can be used as a risk regulator for breast cancer in BRCA gene carriers. Through screening, we focused on 13 genes that are significantly associated with the risk of breast cancer, including IGF1 receptor ligand IGF2 and insulin, IGF binding protein and so on. This study confirmed that IGF2 is a risk regulator (Neuhausen, 2011) associated with breast cancer in BRAC1 and BRCA2 mutations.

4 Outlook

In conclusion, the abnormal activation of IGF2 signaling pathway, the hypomethylation of IGF2 gene and the increase of peripheral free IGF2 level can promote the growth, transcription and expression of breast cancer cells, suggesting that IGF2 gene has potential for diagnosis and prediction of breast cancer. The whole process is accomplished by affecting multiple signal transduction pathways. Therefore, it is of great significance to study the clinical application value and mechanism of IGF2 in breast cancer treatment, promote the development of breast cancer treatment and develop safe and effective anti-cancer drugs.

Authors’ contributions

ZDW read and approved the final manuscript. LWQ and YM wrote and translated the manuscript. LWQ, YSR, ZJY, ZBZ, and YM collected materials. All authors read and approved the final manuscript.

Acknowledgments

This work was supported by the Heilongjiang scientific research project (grants 201810).

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