APOBEC3B Expression Research Articles

APOBEC3B Nuclear Localization Requires Two Distinct N-Terminal Domain Surfaces

The APOBEC3 family of cytosine deaminases catalyzes the conversion of cytosines-to-uracils in single-stranded DNA. Traditionally, these enzymes are associated with antiviral immunity and restriction of DNA-based pathogens. However, a role for these enzymes in tumor evolution and metastatic disease has also become evident. The primary APOBEC3 candidate in cancer mutagenesis is APOBEC3B (A3B) for three reasons: (1) A3B mRNA is upregulated in several different cancers, (2) A3B expression and mutational loads correlate with poor clinical outcomes, and (3) A3B is the only family member known to be constitutively nuclear. Previous studies have mapped non-canonical A3B nuclear localization determinants to a single surface-exposed patch within the N-terminal domain (NTD). Here, we show that A3B has an additional, distinct, surface-exposed NTD region that contributes to nuclear localization. Disruption of residues within the first 30 amino acids of A3B (import surface 1) or loop 5/α-helix 3 (import surface 2) completely abolish nuclear localization. These import determinants also graft into NTDs of related family members and mediate re-localization from cell-wide-to-nucleus or cytoplasm-to-nucleus. These findings demonstrate that both sets of residues are required for non-canonical A3B nuclear localization and describe unique surfaces that may serve as novel therapeutic targets.

APOBEC3B and APOBEC mutational signature as potential predictive markers for immunotherapy response in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is known to carry heavy mutation load. Besides smoking, cytidine deaminase APOBEC3B plays a key role in the mutation process of NSCLC. APOBEC3B is also reported to be upregulated and predicts bad prognosis in NSCLC. However, targeting APOBEC3B high NSCLC is still a big challenge. Here we show that APOBEC3B upregulation is significantly associated with immune gene expression, and APOBEC3B expression positively correlates with known immunotherapy response biomarkers, including: PD-L1 expression and T-cell infiltration in NSCLC. Importantly, APOBEC mutational signature is specifically enriched in NSCLC patients with durable clinical benefit after immunotherapy and APOBEC mutation count can be better than total mutation in predicting immunotherapy response. In together, this work provides evidence that APOBEC3B upregulation and APOBEC mutation count can be used as novel predictive markers in guiding NSCLC checkpoint blockade immunotherapy.

Expression of DNA cytosine deaminase APOBEC3 proteins, a potential source for producing mutations, in gastric, colorectal and prostate cancers.

APOBEC3B is a deaminase that possesses DNA C-to-T editing activity. A recent report showed that APOBEC3B mRNA was overexpressed in breast cancer and that its expression was responsible for the high C-to-T mutation spectrum in breast cancer, suggesting that APOBEC3B could serve as a source for producing mutations. To see whether APOBEC3B is overexpressed in other common cancers at the protein level, we investigated APOBEC3 protein expression in 100 gastric, 103 colorectal and 107 prostate cancer tissues as well as in 10 breast cancers by immunohistochemistry using antibody that could detect APOBEC3B, APOBEC3F and APOBEC3D proteins. In the cancers, APOBEC3 expression was detected in 100% of breast cancers, 67% of gastric, 84% of colorectal and 67% of prostate cancer. Also, it was expressed in 100% of normal breast, 90% of normal stomach, 82% of normal colon and 93% of normal prostate tissues. In contrast to earlier data that showed an increased APOBEC3B expression in breast cancer cells compared to normal breast cells, APOBEC3 expression in cancers was lower than in normal tissues (gastric and prostate cancer) or was not different from normal tissues (colorectal and breast cancer). There was no significant association of APOBEC3 expression with clinocopathological parameters, including histology, metastasis and stage. Our data indicate that APOBEC3 overexpression might not be restricted to specific cancer types. Also, APOBEC3 expression in many normal epithelial cells suggests that there might be a mutation unrelated function of APOBEC3 in the normal cells.

Correlation of gene expression and associated mutation profiles of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT with chemosensitivity of cancer cell lines to drug treatment

BackgroundThe APOBEC gene family of cytidine deaminases plays important roles in DNA repair and mRNA editing. In many cancers, APOBEC3B increases the mutation load, generating clusters of closely spaced, single-strand-specific DNA substitutions with a characteristic hypermutation signature. Some studies also suggested a possible involvement of APOBEC3A, REV1, UNG, and FHIT in molecular processes affecting APOBEC mutagenesis. It is important to understand how mutagenic processes linked to the activity of these genes may affect sensitivity of cancer cells to treatment.ResultsWe used information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources to examine associations of the prevalence of APOBEC-like motifs and mutational loads with expression of APOBEC3A, APOBEC3B, REV1, UNG, and FHIT and with cell line chemosensitivity to 255 antitumor drugs. Among the five genes, APOBEC3B expression levels were bimodally distributed, whereas expression of APOBEC3A, REV1, UNG, and FHIT was unimodally distributed. The majority of the cell lines had low levels of APOBEC3A expression. The strongest correlations of gene expression levels with mutational loads or with measures of prevalence of APOBEC-like motif counts and kataegis clusters were observed for REV1, UNG, and APOBEC3A. Sensitivity or resistance of cell lines to JQ1, palbociclib, bicalutamide, 17-AAG, TAE684, MEK inhibitors refametinib, PD-0325901, and trametinib and a number of other agents was correlated with candidate gene expression levels or with abundance of APOBEC-like motif clusters in specific cancers or across cancer types.ConclusionsWe observed correlations of expression levels of the five candidate genes in cell line models with sensitivity to cancer drug treatment. We also noted suggestive correlations between measures of abundance of APOBEC-like sequence motifs with drug sensitivity in small samples of cell lines from individual cancer categories, which require further validation in larger datasets. Molecular mechanisms underlying the links between the activities of the products of each of the five genes, the resulting mutagenic processes, and sensitivity to each category of antitumor agents require further investigation.

APOBEC3B Signature Mutations Benefit BK Polyomavirus

BK polyomavirus (BKV) causes nephropathy in kidney transplant recipients (KTRs). The virus has also been implicated as a possible cause of bladder and kidney cancers. Two KTRs who developed BKV nephropathy followed by renal carcinoma both showed a swarm of BKV sequence variants encoding non-silent mutations in surface loops of the viral major coat protein. The appearance and disappearance of these mutations over time suggests intra-patient evolution of the virus. Some of the observed mutations conferred resistance to antibody-mediated neutralization. The mutations also modified the spectrum of receptor glycans the virus engages during the infectious entry process. Nearly all observed mutations are consistent with DNA damage caused by APOBEC3B, an antiviral cytosine deaminase. This is intriguing in light of a recent report showing that BKV induces APOBEC3B expression. The results indicate that polyomaviruses can employ APOBEC3B to acquire beneficial site-specific mutations, conceivably with carcinogenic consequences for the host.

Abstract P5-05-04: The APOBEC3B transgenic mouse

Abstract Background: Massively parallel DNA sequencing efforts have facilitated the production of catalogs of the somatic mutations present in cancer genomes. Examining the sequence context of the mutations it is possible to identify patterns that recur frequently. These mutational patterns or “signatures” enable inference into the mutational processes responsible for the patterns. Investigators at the Sanger Institute in the UK have produced a compendium of 30 “Signatures of Mutational Processes in Human Cancer”. Two of these signatures have been attributed to the actions of APOBEC cytidine deaminases. Data from other investigators suggest that APOBEC3B (A3B) is more likely to play a role than are other members of the APOBEC family. The evidence to support the hypothesis that APOBEC activation is responsible for the mutation patterns is limited at present. It is important to establish whether these enzymes play a role in oncogenesis as the APOBEC mutational signature had been noted to be present in 1 of every 6 cancer specimens and second only to aging in prevalence. Methods: In order to determine if overexpression of APOBEC3B in the mammary gland of mice is tumorigenic and produces the mutational signatures attributed to its activity, we contracted with a commercial vendor for the production of an APOBEC3B transgenic mouse on a 129:C57BL/6 background. Of note, in the course of the production of the transgenic, the intron between exons 5 and 6 was retained in the construct to prevent the expression of the highly mutatgenic protein in E. coli. The transgene was placed 3' of a stop sequence flanked by LoxP sites within the ROSA26 locus. Mating with MMTV-Cre mice restricts the excision of the stop cassette and expression of APOBEC3B to the mammary tissue. A3B heterozygotes from the F1 generation were bred to produce A3B homozygotes. Homozygotes have been allowed to undergo multiple pregnancies and have been observed for up to 12 months as of June 2017. Results: 3 mice have developed malignancies. Two mice appear to have developed lymphomas and the epithelial cells of a mammary gland of a third mouse (homozygote) at 11 months of age are diffusely hyperplastic. The cells are arranged in rows ranging from 2-4 cells in thickness. Periodic acid–Schiff (PAS) staining confirms that the basement membrane is intact around the hyperplasic cells. There is no anisocytosis, anisokaryosis, desmoplasia, necrosis or mitotic figures noted in the gland. Conclusions: At 11 month of age, the mammary glands of APOBEC3B transgenic mice are beginning to display histologic abnormalities associated with breast oncogenesis. Observation will continue. With the passage of time we expect to observe more advanced and larger mammary lesions. DNA will be extracted from any invasive mammary tumors, sequenced and the mutation signature determined using the EMu: Expectation-Maximisation inference of mutational signatures software available from Sanger. Citation Format: Choi M, Lyle LT, Song C, Clare SE. The APOBEC3B transgenic mouse [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-04.

APOBEC-mediated mutagenesis in urothelial carcinoma is associated with improved survival, mutations in DNA damage response genes, and immune response.

APOBEC enzymes are responsible for a mutation signature (TCW>T/G) implicated in a wide variety of tumors. We explore the APOBEC mutational signature in bladder cancer and the relationship with specific mutations, molecular subtype, gene expression, and survival using sequencing data from The Cancer Genome Atlas (n = 395), Beijing Genomics Institute (n = 99), and Cancer Cell Line Encyclopedia. Tumors were split into “APOBEC-high” and “APOBEC-low” based on APOBEC enrichment. Patients with APOBEC-high tumors have better overall survival compared to those with APOBEC-low tumors (38.2 vs. 18.5 months, p = 0.005). APOBEC-high tumors are more likely to have mutations in DNA damage response genes (TP53, ATR, BRCA2) and chromatin regulatory genes (ARID1A, MLL, MLL3), while APOBEC-low tumors are more likely to have mutations in FGFR3 and KRAS. APOBEC3A and APOBEC3B expression correlates with mutation burden, regardless of bladder tumor molecular subtype. APOBEC mutagenesis is associated with increased expression of immune signatures, including interferon signaling, and expression of APOBEC3B is increased after stimulation of APOBEC-high bladder cancer cell lines with IFNγ. In summary, APOBEC-high tumors are more likely to have mutations in DNA damage response and chromatin regulatory genes, potentially providing more substrate for APOBEC enzymes, leading to a hypermutational phenotype and the subsequent enhanced immune response.

Ductal cells of minor salivary glands in Sjögren's syndrome express LINE-1 ORF2p and APOBEC3B.

Type I interferon activation is a hallmark event in Sjögren's syndrome. L1 retroelements stimulate plasmacytoid dendritic cells, activating the type I interferons, and are regulated by various mechanisms, including the APOBEC3 deaminases. As L1s are potential trigger factors in autoimmunity, we aimed to investigate the immunohistochemical localization of L1 ORF2p and its inhibitor APOBEC3B protein in minor salivary glands of Sjögren's syndrome patients. Twenty minor salivary gland-tissue samples from 20 Sjögren's syndrome patients, classified according to Tarpley's histological criteria, and 10 controls were evaluated for L1 ORF2p and APOBEC3B expression via immunohistochemistry. L1 ORF2p was expressed in 17/20 SS patients and all controls. APOBEC3B expression was observed in 15/20 Sjögren's syndrome patients, 5/5 chronic sialadenitis, and 3/5 normal minor salivary glands. Both antibodies stained the cytoplasm of the ductal epithelial cells. Negative staining was observed in the acinar cells. L1 ORF2p-positive immunostaining was significantly lower in Tarpley IV Sjögren's syndrome patients than controls (P = .039), and APOBEC3B-positive staining was significantly lower in Tarpley I compared to Tarpley II Sjögren's syndrome patients (P = .008) and controls (P = .035). L1 ORF2p and APOBEC3B are expressed in the ductal epithelial cells of minor salivary glands that are among the key targets in Sjögren's syndrome. L1 ORF2p expression may promote the L1 ability to act as an intrinsic antigen in Sjögren's syndrome. The potential future use of L1 ORF2-reverse transcriptase inhibitors in autoimmunity supports further investigation of L1 epigenetic regulation by APOBEC3 enzymes.

Cytosine Deaminase APOBEC3A Sensitizes Leukemia Cells to Inhibition of the DNA Replication Checkpoint.

Mutational signatures in cancer genomes have implicated the APOBEC3 cytosine deaminases in oncogenesis, possibly offering a therapeutic vulnerability. Elevated APOBEC3B expression has been detected in solid tumors, but expression of APOBEC3A (A3A) in cancer has not been described to date. Here, we report that A3A is highly expressed in subsets of pediatric and adult acute myelogenous leukemia (AML). We modeled A3A expression in the THP1 AML cell line by introducing an inducible A3A gene. A3A expression caused ATR-dependent phosphorylation of Chk1 and cell-cycle arrest, consistent with replication checkpoint activation. Further, replication checkpoint blockade via small-molecule inhibition of ATR kinase in cells expressing A3A led to apoptosis and cell death. Although DNA damage checkpoints are broadly activated in response to A3A activity, synthetic lethality was specific to ATR signaling via Chk1 and did not occur with ATM inhibition. Our findings identify elevation of A3A expression in AML cells, enabling apoptotic sensitivity to inhibitors of the DNA replication checkpoint and suggesting it as a candidate biomarker for ATR inhibitor therapy. Cancer Res; 77(17); 4579-88. ©2017 AACR.

Roles of APOBEC3A and APOBEC3B in Human Papillomavirus Infection and Disease Progression.

The apolipoprotein B messenger RNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family of cytidine deaminases plays an important role in the innate immune response to viral infections by editing viral genomes. However, the cytidine deaminase activity of APOBEC3 enzymes also induces somatic mutations in host genomes, which may drive cancer progression. Recent studies of human papillomavirus (HPV) infection and disease outcome highlight this duality. HPV infection is potently inhibited by one family member, APOBEC3A. Expression of APOBEC3A and APOBEC3B is highly elevated by the HPV oncoproteins E6 and E7 during persistent virus infection and disease progression. Furthermore, there is a high prevalence of APOBEC3A and APOBEC3B mutation signatures in HPV-associated cancers. These findings suggest that induction of an APOBEC3-mediated antiviral response during HPV infection may inadvertently contribute to cancer mutagenesis and virus evolution. Here, we discuss current understanding of APOBEC3A and APOBEC3B biology in HPV restriction, evolution, and associated cancer mutagenesis.

P53 controls expression of the DNA deaminase APOBEC3B to limit its potential mutagenic activity in cancer cells.

Cancer genome sequencing has implicated the cytosine deaminase activity of apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) genes as an important source of mutations in diverse cancers, with APOBEC3B (A3B) expression especially correlated with such cancer mutations. To better understand the processes directing A3B over-expression in cancer, and possible therapeutic avenues for targeting A3B, we have investigated the regulation of A3B gene expression. Here, we show that A3B expression is inversely related to p53 status in different cancer types and demonstrate that this is due to a direct and pivotal role for p53 in repressing A3B expression. This occurs through the induction of p21 (CDKN1A) and the recruitment of the repressive DREAM complex to the A3B gene promoter, such that loss of p53 through mutation, or human papilloma virus-mediated inhibition, prevents recruitment of the complex, thereby causing elevated A3B expression and cytosine deaminase activity in cancer cells. As p53 is frequently mutated in cancer, our findings provide a mechanism by which p53 loss can promote cancer mutagenesis.

The 30 kb deletion in the APOBEC3 cluster decreases APOBEC3A and APOBEC3B expression and creates a transcriptionally active hybrid gene but does not associate with breast cancer in the European population.

APOBEC3B, in addition to other members of the APOBEC3 gene family, has recently been intensively studied due to its identification as a gene whose activation in cancer is responsible for a specific pattern of massively occurring somatic mutations. It was recently shown that a common large deletion in the APOBEC3 cluster (the APOBEC3B deletion) may increase the risk of breast cancer. However, conflicting evidence regarding this association was also reported. In the first step of our study, using different approaches, including an in-house designed multiplex ligation-dependent probe amplification assay, we analyzed the structure of the deletion and showed that although the breakpoints are located in highly homologous regions, which may generate recurrent occurrence of similar but not identical deletions, there is no sign of deletion heterogeneity. This knowledge allowed us to distinguish transcripts of all affected genes, including the highly homologous canonical APOBEC3A and APOBEC3B, and the hybrid APOBEC3A/APOBEC3B gene. We unambiguously confirmed the presence of the hybrid transcript and showed that the APOBEC3B deletion negatively correlates with APOBEC3A and APOBEC3B expression and positively correlates with APOBEC3A/APOBEC3B expression, whose mRNA level is >10-fold and >1500-fold lower than the level of APOBEC3A and APOBEC3B, respectively. In the next step, we performed a large-scale association study in three different cohorts (2972 cases and 3682 controls) and showed no association of the deletion with breast cancer, familial breast cancer or ovarian cancer. Further, we conducted a meta-analysis that confirmed the lack of the association of the deletion with breast cancer in non-Asian populations.

Abstract 2590: Analysis of APOBEC3A and APOBEC3B mutational signatures using next-generation sequencing data from cancer cell lines

Abstract The APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) gene family of cytidine deaminases includes evolutionarily conserved genes that play important roles in DNA repair and mRNA editing. Activity of at least two APOBEC family members, APOBEC3A and APOBEC3B, can lead to kataegis, a mutagenic process in cancer cells that generates clusters of closely spaced, single strand specific C->T DNA substitutions. APOBEC mutagenesis has a characteristic signature, most commonly represented by the 5’-Tp(C->T)pW-3’ sequence motif, with additional substitutions also reported. This hypermutation signature and high mRNA expression of APOBEC3A and APOBEC3B have been associated with several cancer types. Most previous studies of APOBEC signatures have examined tumor sequence data from clinical samples, for which limited or no information about drug response was available. We investigated the presence of the mutational signature and mRNA expression patterns of the APOBEC3A and APOBEC3B genes in extensively characterized cell lines, in order to identify those cell lines that carry mutations generated by kataegis, with the aim of establishing associations between the APOBEC mutational signature, individual cancer types, and the patterns of sensitivity to antitumor agents. For this purpose, we analyzed whole exome sequencing (WES) data and mRNA expression of the APOBEC3A and APOBEC3B genes in two resources with extensive drug response data: the NCI-60 cell line panel, which includes 59 human cancer cell lines representing 9 cancer types and drug response information for thousands of anticancer agents, and the Cancer Cell Line Encyclopedia (CCLE), which provides WES, whole genome, and RNA-seq sequence information on hundreds of cancer cell lines and drug response data to over 200 agents. We analyzed WES data of 325 CCLE cell lines and 59 NCI-60 cell lines, with variants identified using GATK pipeline and Varscan2 software. The variants in each cell line were filtered to remove common polymorphisms in dbSNP and 1000 Genome Project databases. We searched the discovered variants for the presence of APOBEC signatures, 5’-Tp(C->AGT)pN-3’, 5’-Tp(C->AGT)pD-3’, and 5’-Tp(C->AGT)pW-3’ in closely spaced (1000 and 10,000 bp) windows that appeared on the same DNA strand. We will discuss the use of optimal filters for detecting APOBEC mutational signatures and will present the analyses of associations between APOBEC signatures, mutational load of the tumor cell lines, APOBEC gene expression, and chemosensitivity to treatment. These results contribute to additional characterization of available cell lines by providing information about specific mutational signatures in different categories of cancer. Our findings may assist with identifying antitumor agents that would be appropriate for treatment of cancer cells with specific signature patterns generated by APOBEC mutagenesis. Citation Format: Suleyman Vural, Julia Krushkal, Richard Simon. Analysis of APOBEC3A and APOBEC3B mutational signatures using next-generation sequencing data from cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2590. doi:10.1158/1538-7445.AM2017-2590

APOBEC3B and IL-6 form a positive feedback loop in hepatocellular carcinoma cells.

APOBEC3 protein families, a DNA cytidine deaminase, were up-regulated in multiple tumors. However, the relationship between Hepatocellular carcinoma (HCC) and APOBEC3B (A3B) remains unknown. It has been confirmed that interleukin-6 (IL-6) has significant impacts on oncogenesis of HCC. Here, we reported that the expression of IL-6 was substantially up-regulated by A3B in HepG2 cells. A3B induced IL-6 expression through relocating HuR to enhance the IL-6 mRNA stability. Further analysis indicated that IL-6 also increased the expression of A3B through JAK1/STAT3 signaling pathway, which formed a positive feedback to maintain the continuous expression of A3B and IL-6, and thereby promoted the prolonged non-resolving inflammation. Collectively, these findings suggest that A3B is essential for oncogenesis of HCC, and is a potential target for preventive intervention.

Elevated APOBEC3B expression drives a kataegic-like mutation signature and replication stress-related therapeutic vulnerabilities in p53-defective cells

Background:Elevated APOBEC3B expression in tumours correlates with a kataegic pattern of localised hypermutation. We assessed the cellular phenotypes associated with high-level APOBEC3B expression and the influence of p53 status on these phenotypes using an isogenic system.Methods:We used RNA interference of p53 in cells with inducible APOBEC3B and assessed DNA damage response (DDR) biomarkers. The mutational effects of APOBEC3B were assessed using whole-genome sequencing. In vitro small-molecule inhibitor sensitivity profiling was used to identify candidate therapeutic vulnerabilities.Results:Although APOBEC3B expression increased the incorporation of genomic uracil, invoked DDR biomarkers and caused cell cycle arrest, inactivation of p53 circumvented APOBEC3B-induced cell cycle arrest without reversing the increase in genomic uracil or DDR biomarkers. The continued expression of APOBEC3B in p53-defective cells not only caused a kataegic mutational signature but also caused hypersensitivity to small-molecule DDR inhibitors (ATR, CHEK1, CHEK2, PARP, WEE1 inhibitors) as well as cisplatin/ATR inhibitor and ATR/PARP inhibitor combinations.Conclusions:Although loss of p53 might allow tumour cells to tolerate elevated APOBEC3B expression, continued expression of this enzyme might impart a number of therapeutic vulnerabilities upon tumour cells.

B-Myb Induces APOBEC3B Expression Leading to Somatic Mutation in Multiple Cancers

The key signature of cancer genomes is the accumulation of DNA mutations, the most abundant of which is the cytosine-to-thymine (C-to-T) transition that results from cytosine deamination. Analysis of The Cancer Genome Atlas (TCGA) database has demonstrated that this transition is caused mainly by upregulation of the cytosine deaminase APOBEC3B (A3B), but the mechanism has not been completely characterized. We found that B-Myb (encoded by MYBL2) binds the A3B promoter, causing transactivation, and this is responsible for the C-to-T transitions and DNA hypermutation in breast cancer cells. Analysis of TCGA database yielded similar results, supporting that MYBL2 and A3B are upregulated and putatively promote C-to-T transitions in multiple cancer types. Moreover, blockade of EGF receptor with afatinib attenuated B-Myb–A3B signaling, suggesting a clinically relevant means of suppressing mutagenesis. Our results suggest that B-Myb–A3B contributes to DNA damage and could be targeted by inhibiting EGF receptor.

Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor.

The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis.IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1/4 protein, thereby leading to A3B upregulation. Since increased levels of TEAD4 are frequently observed in many cancers, an understanding of the direct link between TEAD and A3B upregulation is of broad oncological interest.

Progressive APOBEC3B mRNA expression in distant breast cancer metastases.

BackgroundAPOBEC3B was recently identified as a gain-of-function enzymatic source of mutagenesis, which may offer novel therapeutic options with molecules that specifically target this enzyme. In primary breast cancer, APOBEC3B mRNA is deregulated in a substantial proportion of cases and its expression is associated with poor prognosis. However, its expression in breast cancer metastases, which are the main causes of breast cancer-related death, remained to be elucidated.Patients and methodsRNA was isolated from 55 primary breast cancers and paired metastases, including regional lymph node (N = 20) and distant metastases (N = 35). APOBEC3B mRNA levels were measured by RT-qPCR. Expression levels of the primary tumors and corresponding metastases were compared, including subgroup analysis by estrogen receptor (ER/ESR1) status.ResultsOverall, APOBEC3B mRNA levels of distant metastases were significantly higher as compared to the corresponding primary breast tumor (P = 0.0015), an effect that was not seen for loco-regional lymph node metastases (P = 0.23). Subgroup analysis by ER-status showed that increased APOBEC3B levels in distant metastases were restricted to metastases arising from ER-positive primary breast cancers (P = 0.002). However, regarding ER-negative primary tumors, only loco-regional lymph node metastases showed increased APOBEC3B expression when compared to the corresponding primary tumor (P = 0.028).ConclusionAPOBEC3B mRNA levels are significantly higher in breast cancer metastases as compared to the corresponding ER-positive primary tumors. This suggests a potential role for APOBEC3B in luminal breast cancer progression, and consequently, a promising role for anti-APOBEC3B therapies in advanced stages of this frequent form of breast cancer.

Aberrantly Expressed APOBEC3B Induces Mutations in Multiple Myeloma

Multiple myeloma (MM) is a malignant plasma cell tumor that arises secondarily from monoclonal gammopathy of uncertain significance (MGUS) due to accumulation of genetic abnormalities. Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC) is a family of DNA cytosine deaminases that play critical roles in innate and acquired immunity. APOBEC proteins catalyze cytosine to uracil deamination and eventually induce C to T mutations in DNA. Recent genome-wide analysis revealed that APOBEC-induced signature DNA mutations accumulate during progression of MM and are associated with poor prognosis (Bolli et. al., Nat Commun. 2014). We previously found that one of the APOBEC3 proteins, APOBEC3B (A3B), induces genomic DNA mutations in a human cell culture model (Shinohara et. al., Sci Rep. 2012) and hypothesized that A3B might induce DNA mutations during onset and progression of MGUS and MM.A deletion polymorphism that removes the entire A3B gene is reported to affect breast cancer prognosis, therefore we first investigated the frequency of A3B deletion allele in 88 Japanese MM/MGUS patients and in healthy controls. 41 patients (46.6%) showed wild type, 42 patients (47.7%) heterozygous, and 5 patients (5.7%) had homozygous deletion allele. We could not detect a significant difference between MM/MGUS patients and healthy controls, suggesting that A3B may not contribute to disease initiation. In contrast, real-time PCR analysis revealed a considerably high expression of A3B mRNA in 23 bone marrow specimens from MM/MGUS patients, except for two samples with homozygous deletion allele. We sorted myeloma cells using anti-CD138 beads and the enriched myeloma cell samples exhibited even higher A3B mRNA expression in 9 MM/MGUS patients (relative quantity to peripheral blood cells [PBMC]: range 1.06 to 55.14, median 7.67).Next, we investigated the APOBEC3 family protein expression profile in six myeloma cell lines: U266, RPMI8226, SKMM1, AMO1, OPM2 and THK72. As expected, all of these cell lines demonstrated remarkably high levels of A3B expression by real-time PCR analysis (relative quantity to PBMC: range 1.22 to 489.4, median 62.47). Western blot analysis using an anti-A3B antibody that we newly generated also confirmed the high A3B protein expression in these myeloma cell lines. In addition, fluorescent immunostaining analysis confirmed high expression of A3B protein localized in the nucleus. Because previous studies showed that A3B expression is activated via the NF-kB signaling pathway, we investigated the regulation of A3B transcription in myeloma cells by p65 knockdown through shRNA lentivirus, resulting in suppression of A3B at protein level.To investigate the mutagenic potential of aberrantly expressed A3B in myeloma cell lines, we evaluated somatic mutations in genomic DNA by differential DNA denaturation PCR (3D-PCR) using stable A3B knock-down cells by shRNA lentivirus simultaneously transduced with a fluorescent gene, mCherry. Interestingly, we detected that scramble shRNA-transduced cells lost fluorescence much faster than those transduced with shRNA against A3B. 3D-PCR amplified mCherry fragments more efficiently at a lower denaturation temperature, indicating that these fragments include more AT-rich sequences. We cloned these DNA fragments and sequenced about 4000 bp in total, and detected prominently more TC > TT mutations in the mCherry sequences from control shRNA-transduced cells (C to T mutation, 3.001%) than in those from A3B shRNA-tranduced cells (C to T mutation, 0.0024%). Additionally, the amplified mCherry sequences from scramble shRNA-transduced cells contained various deletions which may be repaired by microhomology-mediated end joining. Since DNA double strand breaks (DSB) are constitutively activated in myeloma cells, we also examined whether aberrantly expressed A3B can induce DSBs. A3B knockdown in RPMI8226 decreased gH2AX signals by fluorescent immunostaining and Western blot analysis, suggesting that A3B induces DSBs.The present study clearly shows that myeloma cells express aberrantly high levels of A3B and suggests that A3B might play crucial roles in clonal evolution or genomic instability of MM/MGUS albeit A3B may not contribute to initiation of MGUS/MM. Whether A3B accelerates chemoresistance or disease progression remains to be elucidated, however, A3B might be a potential therapeutic target in MM/MGUS. DisclosuresIida:Janssen Pharmaceuticals: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Takaori-Kondo:Mochida Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Pfizer: Research Funding; Kyowa Kirin: Research Funding; Astellas Pharma: Research Funding; Eisai: Research Funding; Alexion Pharmaceuticals: Research Funding; Chugai Pharmaceutical: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Merck Sharp and Dohme: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Shionogi: Research Funding; Toyama Chemical: Research Funding; Cognano: Research Funding.

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