ABOBEC2, osuus tumorigeneesissä pohdittavana

APOBEC2 ja tumorigenesis

Excessive activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide 2 (APOBEC2) contributes to liver and lung tumorigenesis

Shunsuke Okuyama , Hiroyuki Marusawa et al. First published: 05 April 2011

https://doi.org/10.1002/ijc.26114

Tutkijaryhmä selvitti transgeenisellä hiirimallilla APOBEC2 geenin fysiologisia ja patologisia vaikutuksia. Alunperin APOBEC2 oli tunnistettu sytidiinideamidaasina, joka pystyisi editoimaan nukleotideja. He etsivät  nyt  näitä merkkejä mahdollisista muunnoksista DNA- ja RNA- sekvensseissä. Sekvednssianalyyseistä selvisi, että APOBEC2:n konstitutiivinen ilmeneminen maksassa johti merkitsevän korkeafrekvenssisiin nukleotidiemuuntumisiin isäntäsolun DNA- ja RNA-sekvensseissä Eif4g2 ja PTEN-geenien transkripteissä. 

 Transgeenisille hiirille kehittyi maksasolusyöpä. Konstitutiivisen APOBEC2-ilmenemisen vaikutus transgeenisten hiirien keuhkoissa aiheutti myös tuumoreita. Tulehduksellinen sytokiini TNFalfa indusoi ektooppisen APOBEC2-ilmenemän maksasoluissa. Poikkeava APOBEC2-ilmenemä aiheutti nukleotidien muuntumisia tiettyjen kohdegeenien transkripteissä ja voi olla osasyynä ihmisen maksasolusyövän kehittymiseen maksatulehdustietä.

 Genomin koodaavien sekvenssien lukumäärä on rajallinen, mutta DNA- ja RNA -sekvensseihin sisältyvää informaatiota voidaan manipuloida siten, että soluissa tuottuu laaja tuotekirjo. Nämä APOBEC- perheen entsyymit pystyvät muovaamaan nukleotidejä ja siten kiinnittämään DNA- tai RNA-rakenteeseen somaattisen mutaation sytidiinideaminaasi-kyvyllään. APOBEC1 kykyjä on kuvattu jo ( ApoB48 ja neurofibromiinin aikaansaaminen; nämä ovat aivolle tärkeitä tekijöitä). APOBEC3G pystyy hypermutatoimaan viruksia ja on täten antivirustekijä. AID ilmenee itukeskuksen B-soluissa ja indusoi somaattisen hypermutaation ja ihmisen DNA-sekvenssin immunoglobuliinigeenien luokissa vaihteen ja rekombinoitumisen, mikä kohentaa immuunipuolustuksen monipuolisuutta. APOBEC2:n osalta on vähemmn tietoa. Sitä ilmenee yksinomaan sydän ja luustolihaksesa, mutta ei tiedetä onko sillä jokin substraatti ja sen vaikutus nukleotidien muovaamiseenkin on epäselvä.

• Abstract.Apolipoprotein B mRNA editing enzyme catalytic polypeptide 2 (APOBEC2) was originally identified as a member of the cytidine deaminase family with putative nucleotide editing activity. To clarify the physiologic and pathologic roles, and the target nucleotide of APOBEC2, we established an APOBEC2 transgenic mouse model and investigated whether APOBEC2 expression causes nucleotide alterations in host DNA or RNA sequences. Sequence analyses revealed that constitutive expression of APOBEC2 in the liver resulted in significantly high frequencies of nucleotide alterations in the transcripts of eukaryotic translation initiation factor 4 gamma 2 (Eif4g2) and phosphatase and tensin homolog (PTEN) genes. Hepatocellular carcinoma developed in 2 of 20 APOBEC2 transgenic mice at 72 weeks of age. In addition, constitutive APOBEC2 expression caused lung tumors in 7 of 20 transgenic mice analyzed. Together with the fact that the proinflammatory cytokine tumor necrosis factor‐α induces ectopic expression of APOBEC2 in hepatocytes, our findings indicate that aberrant APOBEC2 expression causes nucleotide alterations in the transcripts of the specific target gene and could be involved in the development of human hepatocellular carcinoma through hepatic inflammation.
• The number of coding sequences in the genome is limited, but the genomic information encoded in DNA or RNA sequences can be manipulated to produce a wide range of expression products in cells.
• 1 Apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) family members are nucleotide‐editing enzymes capable of inserting somatic mutations in DNA and/or RNA through their cytidine deaminating activity. 
• 2 The APOBEC family comprises APOBEC1, ‐2, ‐3A, ‐3B, ‐3C, ‐3DE, ‐3F, ‐3G, ‐3H, ‐4, activation‐induced cytidine deaminase (AID) in humans, and APOBEC1, ‐2, ‐3, and AID in mice, and contribute to producing various physiologic outcomes by modifying target gene sequences.
• 3-5 For example, APOBEC1 participates in lipid metabolism by deaminating a specific cytidine to uridine in Apolipoprotein (Apo‐) B transcript sequences. The nucleotide change induced by APOBEC1 activity results in the formation of a termination codon in an Apo‐B48 mRNA, leading to the production of molecules about half the size of a full‐length genomically encoded Apo‐B100.
• 6, 7 APOBEC3G is a cytidine deaminase that induces hypermutation in viral DNA sequences and acts as a host defense factor against various viruses, including HIV‐1 and hepatitis B viruses
• .8-15 On the other hand, AID is expressed in germinal center B‐cells and induces somatic hypermutation and class switch recombination of the immunoglobulin genes encoded in human DNA sequences, resulting in the amplification of immune diversity. 
• 16, 17 APOBEC1, APOBEC3G and AID thus create nucleotide changes in their preferential target DNA or RNA structures.
•  In contrast to these APOBEC proteins, little is known about the function and editing activity of APOBEC2. Although previous reports indicate that murine APOBEC2 mRNA and protein are expressed exclusively in heart and skeletal muscle, the substrate and function of APOBEC2 and whether APOBEC2 has nucleotide editing activity remain unknown18., 19

Accumulating evidence suggests that excessive or aberrant activity of APOBEC family members leads to tumorigenesis through their nucleotide editing of tumor‐related genes. Transgene expression of APOBEC1 causes dysplasia and carcinoma in mouse and rabbit liver due to its aberrant editing of the eukaryotic translation initiation factor 4 gamma 2 (Eif4g2).20, 21 A more striking tumor phenotype is observed in mice with constitutive and ubiquitous AID expression. We previously demonstrated that AID transgenic (Tg) mice developed tumors in various organs, including liver, lung, stomach and lymphoid organs, accompanied by the accumulation of somatic mutations on several tumor‐related genes such as Tp53 and Myc. 22, 23 

 Interestingly, we also found that proinflammatory cytokine stimulation induces a substantial upregulation of APOBEC2 transcription via the activation of the transcriptional factor nuclear factor‐κB (NF‐κB) in hepatoma‐derived cells, whereas only trace amounts of endogenous APOBEC2 expression are detectable in normal hepatocytes.24 On the basis of the fact that most human hepatocellular carcinoma (HCC) arises in the setting of chronic liver disease with the features of chronic hepatitis or liver cirrhosis, we hypothesized that APOBEC2 enzyme activity has a role in the accumulation of genetic alterations in tumor‐related genes under conditions of hepatic inflammation, thereby contributing to the development of HCC. In this study, we investigated the putative nucleotide editing ability of APOBEC2 on the host genes in hepatocytes, and its relevance to carcinogenesis by establishing Tg mice that constitutively express APOBEC2. 

 

Abbreviations

APOBEC: Apolipoprotein B mRNA editing enzyme catalytic polypeptide;

EIF4G2: Eukaryotic translation initiation factor 4 gamma 2;

AID: activation‐induced cytidine deaminase;

Apo‐: Apolipoprotein;

Tg: transgenic;

NF‐κB: nuclear factor‐κB;

HCC: hepatocellular carcinoma;

TNF: tumor necrosis factor;

cDNA: Complementary DNA;

RT‐PCR: real‐time reverse‐transcription polymerase chain reaction;

ER: estrogen receptor 

Results

Detection of endogenous APOBEC2 protein expression in hepatocytes

We previously reported that transcription of APOBEC2 is induced by the proinflammatory cytokine tumor necrosis factor (TNF)‐α through the activation of NF‐κB. To confirm whether endogenous APOBEC2 protein is elevated in response to TNF‐α stimulation in human hepatocytes, we generated a rabbit polyclonal antibody against a common amino‐acid sequence to human and murine APOBEC2. Using this anti‐APOBEC2 antibody, we first confirmed that plasmid‐derived exogenous APOBEC2 protein was efficiently detected by immunoblotting analysis (Fig. 1a). We then examined whether endogenous APOBEC2 protein was upregulated by TNF‐α stimulation in Huh‐7 cells. Immunoblotting analysis using the APOBEC2 antibody revealed that endogenous APOBEC2 protein expression was strongly induced after TNF‐α stimulation, suggesting that APOBEC2 protein has a role in hepatocyte function under inflammatory conditions (Fig. 1b). 

Discussion

Among the APOBEC family members, APOBEC2 and AID homologs can be traced back to bony fish, whereas APOBEC1 and APOBEC3s are restricted to mammals.30, 31 The broad preservation of the APOBEC2 homolog among vertebrates suggests that APOBEC2 has a critical role in the physiology of many species. Little is currently known, however, about the biologic activity of APOBEC2 in any type of cells. Moreover, it is not known whether APOBEC2 possesses nucleotide editing activities like other APOBEC family member proteins.

 In the present study, we demonstrated for the first time that APOBEC2 expression triggered nucleotide alterations in RNA sequences of the specific genes in hepatocytes. In addition, our findings suggest that APOBEC2 could contribute to tumorigenesis via the nucleotide alterations of RNA sequences of the target genes. 

On the basis of the close sequence homology of APOBEC2 with other APOBEC proteins, APOBEC2 is thought to exhibit deamination activity to achieve nucleotide editing. Indeed, crystal structure analysis indicates that APOBEC2 contains amino acid residues with 4 monomers in each asymmetric unit that form a tetramer with an atypical elongated shape, and this prominent feature of the APOBEC2 tetramer suggests that the active sites are accessible to large RNA or DNA substrates.32 In the present study, in a mouse model with constitutive APOBEC2 expression, nucleotide alterations were induced in RNA sequences of the Eif4g2 and possibly the Pten genes in hepatocytes. Similar to its effect in vivo, aberrant APOBEC2 expression in cultured hepatocyte‐derived cells induced nucleotide alterations in the EIF4G2 transcripts. Although our findings demonstrate potential mutator activity of the APOBEC2 protein, it is unclear why the EIF4G2 transcripts were more sensitive to APOBEC2 activity than other genes in hepatocytes. APOBEC1 expression in hepatocytes also induced somatic mutations in the transcripts of the EIF4G2 gene.21 Thus, the sequences of the EIF4G2 gene might be a common target for the nucleotide editing effects of both the APOBEC1 and APOBEC2 proteins.

Further analysis is required to identify the specific target genes of APOBEC2‐mediated nucleotide editing in hepatocytes.

An intriguing finding was that the mouse model with constitutive and ubiquitous APOBEC2 expression spontaneously developed epithelial neoplasia in the lung and liver tissues as well as lymphoma. Similar phenotypic findings are observed in mouse models expressing APOBEC1 or AID. Tg mice with RNA‐editing enzyme APOBEC1 expression develop HCC at high frequencies with an accumulation of somatic mutations at multiple sites on Eif4g2 mRNA.20, 21 We also demonstrated that AID Tg mice develop tumors in several organs, including the liver, lung, stomach, and the lymphoid tissues through the accumulation of genetic changes induced by the genotoxic effect of AID.22, 23, 28  

The molecular mechanisms underlying the contribution of constitutive APOBEC2 expression to tumorigenesis remain unknown.  

The number of mRNA mutations observed in the Eif4g2 and Pten genes in the liver of APOBEC2 Tg mice suggests that these genetic alterations by APOBEC2 have a role in the development of HCC. Indeed, the EIF4G2 gene is a candidate molecule responsible for oncogenesis caused by the overexpression of APOBEC1,21 and is frequently downregulated in human cancer tissues.33 In addition, PTEN is one of the most frequently mutated tumor‐suppressor genes in human cancers.34 Thus, the tumorigenesis caused by constitutive APOBEC2 expression might be a consequence of promiscuous nucleotide editing. 

Recent studies revealed that the expression of a subset of APOBEC family members is induced by cytokine stimulation in liver tissues.

 For example, we and other investigators demonstrated that APOBEC3G expression is triggered by interferon‐α in hepatocytes, suggesting that APOBEC3G acts as a host defense in response to interferon signaling against viral infection.35-37

  In this study, we showed that TNF‐α induced APOBEC2 protein expression in human hepatocytes. Considering the fact that chronic inflammation has important roles in human HCC development,38, 39 the finding that APOBEC2 is induced by proinflammatory cytokine stimulation and induces nucleotide alterations in tumor‐related genes in hepatocytes provides a novel idea that aberrant expression of APOBEC2 in epithelial cells acts as a genotoxic factor linking inflammation and cancer development.  

The tumorigenic phenotype of the APOBEC2‐Tg mice further suggests that APOBEC2 is involved in carcinogenesis of the liver tissue under conditions of chronic inflammation, the typical procancerous background of human HCC. 

In conclusion, our findings provide the first direct evidence that APOBEC2 induces nucleotide changes preferentially in the Eif4g2 and possibly the Pten genes, and the constitutive expression of APOBEC2 in epithelial tissues contributes to the development of various tumors including HCC and lung cancers. Understanding the pathologic role of APOBEC2 provides new insight into the mechanisms of cancer development in the liver underlying chronic inflammation. During our manuscript preparation, Sato et al. reported that they could not find the evidence of APOBEC2's affinity for RNA or high‐stoichiometry association with a partner which usually associated with the known RNA editing enzymes.40 Thus, further analyses would be required to clarify whether APOBEC2 dose possess an RNA‐editing activity against specific target genes or overexpression of APOBEC2 causes nucleotide alterations in genome senquences in a promiscuous manner in hepatocytes.

Supporting Information

References

Citing Literature