|Year : 2018 | Volume
| Issue : 2 | Page : 37-44
Aberrant expression of p14ARF in human cancers: A new biomarker?
Kazushi Inoue, Elizabeth A Fry
Department of Pathology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA
|Date of Web Publication||4-Feb-2019|
Department of Pathology, Wake Forest University Health Sciences, Winston-Salem, NC 27157
Source of Support: None, Conflict of Interest: None
The ARF and INK4a genes are located on the same CDKN2a locus, both showing its tumor-suppressive activity. ARF has been shown to monitor potentially harmful oncogenic signalings, making incipient cancer cells undergo senescence or programmed cell death to prevent cancer. On the other hand, INK4a detects both aging and incipient cancer cell signals. The efficiency of detection of oncogenic signals is more efficient for the for the former than the latter in the mouse system. Both ARF and INK4a genes are inactivated by gene deletion, promoter methylation, frameshift, aberrant splicing although point mutations for the coding region affect only the latter. Recent studies show the splicing alterations that affect only ARF or both ARF and INK4a genes, suggesting that ARF is inactivated in human tumors more frequently than what was previously thought. The ARF gene is activated by E2Fs and Dmp1 transcription factors while it is repressed by Bmi1, Tbx2/3, Twist1, and Pokemon nuclear proteins. It is also regulated at protein levels by Arf ubiquitin ligase named ULF, MKRN1, and Siva1. The prognostic value of ARF overexpression is controversial since it is induced in early-stage cancer cells to eliminate premalignant cells (better prognosis); however, it may also indicate that the tumor cells have mutant p53 associated with worse prognosis. The ARF tumor suppressive protein can be used as a biomarker to detect early-stage cancer cells as well as advanced stage tumors with p53 inactivation.
Keywords: ARF, cancer, expression, INK4a, prognosis
|How to cite this article:|
Inoue K, Fry EA. Aberrant expression of p14ARF in human cancers: A new biomarker?. Tumor Microenviron 2018;1:37-44
| Introduction|| |
Since the cloning of the alternate reading frame of the mouse Arf/Ink4a locus, the Arf tumor suppressor has been reported to be a sensor for hyperproliferative stimuli stemming from mutant Ras and c-Myc oncoproteins.,, p19Arf (p14ARF in humans) and p16Ink4a mRNAs are generated from separate and first exons 1β and 1α (19.4 kilobase pairs [kb] apart in humans) which splice into two common exons 2 and 3 [Figure 1]. These two genes are different tumor suppressor since p19Arf uses only exons 1 and 2 (also p14ARF) while p16Ink4a uses all of the exons 1–3 for production of the protein., This locus has a unique genomic structure not found in other mammalian genes due to the splicing used by Arf which uses an alternate reading frame in the coding region of exon two. Of note, this ARF-INK4a (CDKN2a) locus is located 11.5 kbp downstream of genomic loci for CDKN2b that encodes for p15INK4b [Figure 1]. The aberrant transcripts from this locus have been described and reviewed. Since RB is regulated by p16INK4a and p53 is regulated by p14ARF, the ARF/INK4a locus is frequently inactivated in human cancers second only to p53 in frequency. Both p19Arf and p16Imk4a act as tumor suppressors in mice,, despite a lack of amino acid sequence similarity. ARF is a highly basic, insoluble protein (pI 11).,, Although human and mouse ARF differ in size (mouse 19 kDa, human 14 kDa) and show only 49% amino acid sequence identity, the functions of the ARF proteins are the same between the two species. The Arf gene exists in other mammals, such as rat, opossum, pig, hamster, and chicken. Ectopic Arf arrests immortal rodent cell lines such as NIH 3T3 as well as transformed human cells,, a classic phenotype of tumor suppressor genes. The main function of ARF is to quench oncogenic signals stemming from hyperproliferation, diverting it to the p53-dependent cell cycle arrest or apoptosis.,, The ability of Arf to inhibit cell cycle progression in a number of cell types suggested that Arf has powerful growth-inhibitory functions in cells, which stimulated researchers to study the in vivo activity of Arf to prevent tumors. Arf sequesters MDM2 in the nucleolus, thus preventing p53 degradation. In addition, it inhibits the transcription factor E2F activity. These activities lead to cell cycle arrest at G1 and G2. Itahana and Zhang in their study reported that the mitochondrial protein p32/C1QBP binds the ARF C-terminus where p32 is required for ARF to localize to mitochondria to induce apoptosis, revealing the essential role of ARF in tumor suppression and programmed cell death. Although it has been believed that most of the tumor-suppressive function of Arf is mediated by p53, accumulating evidence has pointed out additional p53-independent functions of Arf through interaction with proteins such as E2Fs, c-Myc, Nuclear factor-kappa B, HIF1α (transcription factors), nucleophosmin (NPM), and ribosomal RNAs (reviewed in 10). A recent study suggests that nuclear factor E2-related factor 2 (NRF2) is a major target of ARF in p53-independent tumor suppression.
|Figure 1: The structure for the human p15INK4b-p14ARF-p16INK4alocus. The genomic structure is well-conserved between human and mice, and thus gene knockout studies have been extensively conducted in mice. The distance between exon 1 β and exon 1α is 19.4 kbp in humans and 12.4 kbp in mice. The exon 1α is 3.8 kbp upstream of exon 2 in humans; 5.2 kbp in mice (from 5' of exon 1α to 5' of exon 2). The ARF-INK4a (CDKN2a) locus is located 11.5 kbp apart from the genomic locus for CDKN2b that encodes for p15INK4b in humans (from 3' of exon 2 for p15INK4bto 5' of exon 1 β). All of p15Ink4b, p19Arf, and p16Ink4agenes act as tumor suppressors as reported by Krimpenfort et al., The DMP1 consensus is located-2.3 kb and-0.31 kb of ARF (shown in red reverse triangles) and-4.04 kb and-1.40 kb of INK4a (pink reverse triangles) in humans. Both of these are Dmp1 target genes although the mode of regulation is different. Pasmant et al. identified a new large antisense noncoding RNA (named ANRIL) to this genomic locus, with a first exon located in the promoter of the p14ARFgene and overlapping the two exons for p15CDKN2b. Expression of ANRIL was simultaneously found with p14ARF both in physiologic and pathologic conditions. Kobayashi et al. found that that p14ARF regulates the stability of the p16INK4a protein in human and mouse cells. Importantly, ARF promoted rapid degradation of p16INK4a protein, which was mediated by the proteasome and more specifically, by interaction of ARF with one of its subunits, regenerating islet-derived protein 3γ. Thus there is a significant crosstalk between ARF and INK4a at the protein level. ULF, MKRN1, and Siva1 are E3 ligases for ARF that accelerates its degradation.,,,,|
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p16INK4a promotes cellular senescence in response to stressors, such as telomere erosion, stalled replication forks, reactive oxygen species, and oncogene activation (reviewed in 14–16). Expression of p16INK4a in normal cells is almost undetectable, but once induced, p16INK4a binds to cyclin-dependent kinase 4/6 (CDK4/6) to inhibit its activity, thereby promoting retinoblastoma (RB)-dependent cell cycle arrest. This tumor-suppressive mechanism limits the growth of early-stage cancer cells, and accordingly, the p16INK4a-CDK4/6-RB axis is disrupted in most human cancers, with inactivation of p16INK4a the most common mechanism.,, Although the induction of p16INK4a by oncogenic stimuli results in a beneficial effect to eliminate incipient cancer cells, it also accelerates cell aging.,, This review is focused on the mechanism of regulation of ARF and its expression in human cancers. Special interests have been put on the capacity of ARF for early-stage tumor detection as well as advanced cancers with the p53 mutation for potential clinical applications since it is not expressed in normal cells.
| Transcriptional Regulation of Arf|| |
Transcriptional control is the major mechanism of Arf regulation since it has a long half-life of 6 h, much longer than those of cyclins. A number of transcription factors regulate Arf, either positively or negatively [Figure 1]. The E2F1 transcription factor induces Arf by directly binding to its consensus sequences, and also the Sp1 sites which are located in the upstream of the exon 1β, thus activating cell growth arrest or apoptosis to avoid the emergence of incipient cancer cells.,, E2F2 and E2F3a also transactivate the Arf promoter. Conversely, E2F3b, a splice isoform of E2F3a that is not regulated by E2Fs, represses Arf transcription and stimulates cellular growth., The observation that the loss of Arf can rescue E2F3b depletion-mediated cell cycle arrest suggests an anti-reciprocal correlation between these two proteins. To prevent hyperproliferation of cells with oncogenic stress, c-Myc activates fail-safe programs such as apoptosis and cellular senescence by inducing Arf transcription. Thus, Myc-induced Arf activates the p53 signaling, preventing immortalization of murine embryonic fibroblasts (MEFs). Bouchard et al. in their study reported that c-Myc signaling increases nuclear FoxO, which, in turn, binds to the Arf promoter to suppress c-Myc-driven lymphomagenesis.
A study by Qi et al. showed that Arf inhibits c-Myc through physical interaction independent of p53. c-Myc increases Arf, which in turn binds to c-Myc to block its ability to activate transcription, induce hyperproliferation, and transformation. Conversely, c-Myc's ability to repress transcription is not influenced by Arf, but rather c-Myc-mediated cell death is enhanced. This feedback represents a p53-independent mechanism to prevent c-Myc-mediated tumor development. The differential effects of Arf on c-Myc function indicate that independent molecular mechanisms mediate c-Myc-induced hyperproliferation and programmed cell death. The same group later showed that Egr1 mediates p53-independent c-Myc-induced apoptosis through an Arf-dependent mechanism. Therefore, c-Myc-Arf-binding switches the intrinsic activity of c-Myc from a proliferative to apoptotic protein independent of p53.
A cyclin D-binding Myb-like protein Dmp1 (also named Dmtf1) also transactivates the Arf promoter. Inoue et al. revealed that Dmp1 (Dmp1α) binds to the Ets consensus (5'-CCCGGATGC-3') of the mouse Arf promoter to activate its transcription in normal MEFs, which results in Arf/p53-dependent cell cycle arrest,,,,,,,,,,,,,, (Dmp1 reviewed in Refs.,,,,). DMP1α transactivates the human ARF promoter, the activity of which is antagonized its splice variant DMP1 β., E2F1 collaborates the Dmp1 to activate the Arf promoter to avoid neoplastic transformation of early-stage cancer cells. Similar mechanisms should be present in human ARF promoter since both E2F and DMP1-consensus sequences are found in the human version.
Arf is actively involved in TGFβ signaling. Zheng et al. in their study reported that TGFβ increases the Arf mRNA levels through Smad2/3 and p38MAPKs in MEFs. Chromatin immunoprecipitation showed that TGFβ stimulates Smad2/3 binding and histone H3 acetylation 5' to exon 1β, followed by increased Arf transcription and tumor suppression. Then the same group showed that TGFβ-mediated induction of Arf correlated with decreased DNA-binding of C/EBPβ to the Arf promoter. Their results indicate that Smad2/3 and C/EBPβ are positive and negative regulators for Arf transcription affected by TGFβ.
Both human and mouse ARF promoters have binding sites for acute myeloid leukemia-1 (AML1), which causes cellular senescence in MEFs. Interestingly, the t (8;21) fusion protein AML1-ETO is frequently expressed in acute leukemia with low ARF expression. Consistently, Shikami et al. showed that the mRNA expression of p14ARF in t (8;21) AML cells was found to be lower than those without t (8;21) translocation. Since p14ARF has been shown to inhibit p53 degradation by binding to MDM2,,, repression of p14ARF expression in t (8;21) AML accelerates the degradation of p53 by MDM2 explaining why genotoxic damage caused by ionizing radiation does not induce p53 response in t (8;21) AML cells.
Repressors of Arf transcription have also been reported [Figure 2]. The polycomb group gene Bmi1-deficient MEFs shows decreased cell cycle progression and increased premature senescence, which are rescued by Arf/Ink4a depletion. Bmi1 requires the EZH2-containing Polycomb-Repressive Complex 2 (PRC2) to repress ARF/INK4a transcription. TBX2 immortalizes MEFs and decreases senescence in normal human cells by repression of Arf transcription [Figure 2]. The basic helix-loop-helix transcription factor Twist1 activates the recruitment of EZH2 to the Arf transcription start site. Thus, it increases the levels of H3K27Me3 on the ARF/INK4a locus, followed by repression of Arf transcription. An intriguing regulator for ARF/INK4a is the long non-coding RNA ANRIL (antisense noncoding RNA in the INK4 locus) [Figure 1], which is transcribed in the antisense orientation.,,ANRIL promotes the epigenetic repression of its tumor suppressors in the ARF/INK4a locus by physically interacting with the Polycomb proteins Suz12 and Cbx7, which in turn recruit the Polycomb repressive complex 1 and 2 to the locus, leading to histone modification and silencing of the locus.
|Figure 2: Oncogenic and tumor suppressive signaling pathways involving Dmp1. Arf is induced by potentially oncogenic signals stemming from overexpression of oncogenes such as c-Myc, E2F1, and activated Ras, which quenches inappropriate mitogenic signaling by diverting incipient cancer cells to undergo p53-dependent growth arrest or cell death.,, Positive input signals for Arf have been shown in red (our own research) or pink (research from other labs). Conversely, negative signals have been shown in T in black. The output signals for Arf have been shown in striped arrows. Both Dmp1-/- and Dmp1+/- mice show hypersensitivity to develop tumors in response to carcinogen or γ-irradiation., D-type cyclins inhibit Dmp1's activity in a Cdk-independent fashion in promoters lacking E2F sites; however, it cooperates with Dmp1α to activate the Ink4a and Arf promoters, to eliminate incipient tumor cells. The Dmp1 promoter is activated by the oncogenic Ras-Raf-Mek-Erk-Jun and HER2-Pi3k-Akt-NF-κB pathways, and thus Ras or HER2-driven carcinogenesis is accelerated in Dmp1-deficient mice.,, The human DMP1 locus generates three splice variants, namely DMP1 α, β, and γ with antagonizing activity between DMP1α and β. DMP1 β and γ transcripts have not been reported in mice. Dmp1α physically interacts with the epigenetic modifier YY1 that affects EZH2 activity. YY1 binds to HDM2 and Dmp1α to accelerate HDM2-mediated polyubiquitination of p53. Our study shows that Dmp1α physically interacts with p53 through p53's carboxyl-terminal and Dmp1's DNA-binding domain. Dmp1α antagonized p53's ubiquitination by HDM2 both in vitro and in cell and restored p53's nuclear localization that had been lost with HDM2 expression; Dmp1 also stabilized p53 binding to transcriptional target genes. Dmp1α-p53 interaction increases the levels of p53 independent of Dmp1's DNA-binding, and hence both p21Cip1and Bbc3 promoters were synergistically activated by co-expression of Dmp1α and p53 in p53-/-; Arf-/-cells. In accordance, the induction of p21Cip1and Bbc3 by genotoxic drug treatment was more seriously affected in Dmp1-/- and p53-/- tissues than in Arf-/-. In summary, Dmp1α stimulates the p53 pathway by direct transactivation of the Arf promoter in response to oncogenic stresses,,, and direct physical interaction with p53 in DNA damage response (DDR)., Mekk1 is activated by a variety of oxidative stress signaling, such as dsDNA breaks, UV, cytokines, osmotic stress, and oncogenes. Activation of MEKK1 by c-Abl in DDR has been reported. MEKK1 is cleaved by caspase 3 following DNA damage to generate ΔMEKK1, which increases the Dmp1α protein by phosphorylation., Loss of PTEN is found in 70% of advanced prostate cancer (PCa), resulting in activation of the Pi3k-Akt pathway that promotes survival by inhibiting apoptosis and causing genomic instability. The tumor suppressor Pten accelerates the conversion of Pip3 to Pip2, and thus is a negative regulator of Pi3k signaling pathway. In PCa, loss of PTEN drives cell cycle arrest and senescence as a tumor suppressive mechanism mediated by upregulation of p53 expression. Accumulating studies show that RNA splicing is affected by DDR, and also roles of YY1 and PTEN in DDR.|
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In general, the relative importance of the Arf gene far exceeds that of p16Ink4a for tumor suppression in mice reflecting the stronger tumor-prone tendency of Arf-deficient mice, than p16Ink4a-deficient mice, leaving an open question in human cases since in vivo assays are not possible in the latter.
| Regulation of Arf at Protein Levels|| |
Although the importance of Arf in cellular senescence and tumor suppression has been well-documented, Arf is also regulated by posttranslational mechanisms. A report from the Sherr's laboratory suggested that Arf protein, although it lacks lysine sites, is polyubiquitinated at its N-terminus followed by proteasomal degradation by an unknown E3-ubiquitin ligase. Then, a number of ubiquitin ligases have been isolated that regulate Arf at the protein level. In short, both transcriptional and posttranscriptional controls are important in Arf regulation.
In 2005, Chen et al. identified a ubiquitin ligase for p53, ARF-BP1/Mule, as a factor associated with p14ARFin vivo. ARF-BP1 has homology to the E6-AP carboxyl terminus motif, and its ubiquitin ligase activity for p53 was inhibited by ARF. ARF-BP1 directly bound and ubiquitinated p53. They also reported that inactivation of endogenous ARF-BP1 was critical for ARF-mediated p53 stabilization. Thus, ARF-BP1 is a crucial mediator for both the p53-dependent and p53-independent functions of the ARF tumor suppressor. In 2010, the same group reported the first E3-ubiquitin ligase for ARF named ULF (ubiquitin ligase for Arf: ULF)[Figure 1]. ULF caused polyubiquitination and proteasomal degradation of ARF, thus activating cell proliferation. ULF interacted with ARF both in vitro and in vivo and promoted lysine-independent ubiquitination and degradation. Consistently, ULF knockdown stabilized ARF in normal human cells, triggering p53-mediated growth arrest.
Chio et al. in their study reported that tumor necrosis factor receptor-associated death domain shuttled dynamically from the cytoplasm into the nucleus to inhibit the interaction between Arf and ULF, thereby promoting Arf protein stability and tumor suppression. Arf stability control is crucial for differentiating normal (low expression) versus oncogenic (high expression) levels of c-Myc, suggesting that the differential effects on ULF-mediated Arf ubiquitination by c-Myc levels act as a checkpoint in oncogene-induced stress response. Very recently, it was reported that the glioma tumor suppressor candidate region gene 2 (GLTSCR2) bound to ARF. The complexes were released to the nucleoplasm where GLTSCR2 increased the binding affinity of ARF for ULF and enhanced degradation by polyubiquitination. Thus, GLTSCR2 is a strong candidate for promoting the subcellular localization and protein stability of ARF.
NPM is a multifunctional protein that forms a stable complex with ARF in the nucleolus. Importantly, NPM protects ARF from proteasome-mediated degradation. NPM and c-Myc, both of which are commonly overexpressed in cancer cells, promoted ARF stabilization by inhibiting ULF-mediated ubiquitination., NPM is mutated in about one-third of primary samples from AML, leading to cytoplasmic dislocation of the protein. Cytoplasmic NPM mutants found in tumor cells do not retain ARF in the nucleolus, and thus unable to stabilize the protein, compromising the activation of the ARF-p53 pathway. Consistently, steady levels of both Arf and p53 are very low in human AML cells expressing cytoplasmic NPM. ULF knockdown stabilized ARF and reactivated p53 responses in AML cells, suggesting that ULF is a bone fide E3 ligase for ARF. They even suggested a novel therapeutic for AML by inhibiting ULF.
Ko et al. reported the second E3-ubiquitin ligase, Makorin 1 (MKRN1), which targets Arf [Figure 1]. MKRN1-deficient MEFs showed decreased cell growth with a simultaneous increase in Arf protein levels. Consistent with these findings, MKRN1 was shown to induce ubiquitination and proteasomal degradation of Arf.
Siva1, the third E3-ubiquitin ligase for ARF, induces the proteasomal degradation of ARF thus inhibiting p53 function, [Figure 1]. Through direct interaction, Siva1 promoted the ubiquitination and degradation of ARF, which in turn, affected the stability of p53. Functionally, Siva1 regulated cell cycle progression and cell proliferation in an ARF/p53-dependent manner. Together, these findings suggest that the mechanisms of ARF regulation at the protein level are critically important in its responses to oncogenic stress.
| Decreased Expression of ARF in Human Cancer|| |
Consistent with the findings in gene knock-out mice, point mutation, promoter methylation, and/or deletion of the ARF/INK4a locus in human cancers have been reported, [Table 1]. However, it is difficult to evaluate the relative importance of p16INK4a and p14ARF individually since mutations or deletions at the ARF/INK4a locus frequently affects both proteins. In general, exon 2 is the site of point mutation, affecting mainly p16INK4a. Point mutations that cause gene frameshift affect both genes. Some of these exon 2 mutations change ARF localization and affect its regulation of downstream target proteins., Silencing of the ARF gene promoter through hypermethylation is frequently observed in human cancers although the frequency was eight times lower in ARF than INK4a in non-small cell lung carcinomas. Simultaneous methylation of both ARF and INK4a is a common occurrence in samples from the accelerated phase of chronic myeloid leukemia.
|Table 1: Mechanisms of gene inactivation of the ARF-INK4a locus. These tumor suppressor genes are either underexpressed or overexpressed in human cancers, which will cause dysfunction. ARF overexpression can reflect the p53 inactivation of the tumor, suggesting the role for detection of tumor cells with p53 mutation.|
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Although it has been shown that p16INK4a is the major player in human tumor suppression, accumulating pieces of information suggest that p14ARF plays a significant role in tumor suppression. In one study, loss of p14ARF expression was found in 38/50 glioblastomas, with 29 showing either homozygous deletion or hypermethylation;ARF was specifically deleted in nine of the samples. An exon 1β mutation that altered the growth-inhibitory properties and intracellular localization of human p14ARF was reported in a melanoma. A germline deletion of exon 1 β has been reported in patients for a family predisposed to malignant melanoma with/without INK4a involvement., Splicing mutations that affect both ARF and INK4a have been reported;,, a splicing error that affects ARF, but not INK4a has also been reported. More recently, the ARF-INK4a hybrid transcript (chARF) has been reported in a melanoma cell line: the fusion protein has ARF activity, but not that of INK4a.ARF haploinsufficiency due to a germline mutation in exon 1β was observed in a family of three individuals with melanoma or breast cancer, which had been proved in a mouse model.
| ARF Overexpression in Human Cancer|| |
Silva et al. analyzed inactivation mechanisms with 100 primary breast carcinomas. RT-PCR showed altered expression of the p14ARF transcript, with 17% overexpression and 26% decreased expression. No detectable alterations were found in cases with overexpression, but 77% of tumors with the decreased expression for p14ARF showed one or more genetic alterations [Table 1]. A correlation was found between decreased p14ARF expression and poor prognostic parameters. They reported that promoter hypermethylation was present in 31% for 16INK4a and 50% for p14ARF in tumors with decreased expression. In conclusion, these tumor suppressors are often co-altered in human breast cancer.
The prognostic value of p14ARF expression was studied in HPV16(+) squamous cell carcinoma of the oropharynx (SCCOP). They determined the HPV16 status and expression of p14ARF and p53 in 552 patients. Patients having rs3731217 TG/GG variant genotype for ARF polymorphism (high ARF) were two to three times more likely to have HPV16(+) compared with tumors with homozygous TT genotype (low ARF). Thus, p14ARF expression predicts HPV16(+) SCCOP tumor patients with longer survival associated with better response to therapy.
Both of BMI1 and EZH2 are epigenetic modifiers of the polycomb group. However, EZH2 overexpression correlates with a poor prognosis in breast cancer while BMI1 overexpression correlates with a good outcome. A study by Pietersen et al. found that INK4a/ARF was expressed in those with EZH2 overexpression, but not with BMI1. They found a significantly higher proportion of p53 mutations in tumors with high EZH2, which will explain why those with high EZH2 respond poorly to therapy. In conclusion, the prognostic value for ARF should be determined simultaneously with the p53 status because ARF is overexpressed in p53 mutant tumors.
| Future Directions|| |
We have reviewed ARF alterations in cancer. ARF is inactivated in human cancer by gene deletion, frameshift, promoter methylation, and point mutations that affect gene splicing. The prognostic significance for ARF inactivation in cancer is controversial: LOH for the ARF/INK4a locus does not affect the survival of breast cancer patients. Although it is possible that tumors with ARF expression have better prognosis due to accelerated removal of incipient cancer cells, overexpression of ARF is also an indicator for p53 mutation, which is associated with increased tumor invasion/metastasis, increased angiogenesis, and resistance to chemo/radio therapy. The prognosis of such patients is poor. Prognostic studies that simultaneously quantitate 14ARF and p53 expression are lacking; it is thus extremely important to conduct clinical studies by quantitating ARF and p53 levels in the same sample. p14ARF can be used as a biomarker to detect cancer cells since it is not expressed in non-transformed cells.
Recent studies suggest that ARF-induced, p53-independent tumor suppression is significantly abrogated by NRF2 overexpression. Thus, NRF2 is a major target of p53-independent tumor suppression by ARF; it also suggests that the ARF-NRF2 interaction acts as a new checkpoint for oxidative stress responses. Extensive studies should be done in this field since p53 is mutated in half of the human cancers.
We would like to thank all other members of Dr. Inoue's laboratory for sharing unpublished research data.
Financial support and sponsorship
Kazushi Inoue was supported by NIH/NCI 2R01CA106314, ACS RSG-07-207-01-MGO, and KG080179.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Maggi LB Jr., Winkeler CL, Miceli AP, Apicelli AJ, Brady SN, Kuchenreuther MJ, et al.
ARF tumor suppression in the nucleolus. Biochim Biophys Acta 2014;1842:831-9.
Basu S, Murphy ME. Genetic modifiers of the p53 pathway. Cold Spring Harb Perspect Med 2016;6:a026302.
Carrasco-Garcia E, Moreno M, Moreno-Cugnon L, Matheu A. Increased arf/p53 activity in stem cells, aging and cancer. Aging Cell 2017;16:219-25.
Quelle DE, Zindy F, Ashmun RA, Sherr CJ. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 1995;83:993-1000.
Quelle DE, Cheng M, Ashmun RA, Sherr CJ. Cancer-associated mutations at the INK4a locus cancel cell cycle arrest by p16INK4a but not by the alternative reading frame protein p19ARF. Proc Natl Acad Sci U S A 1997;94:669-73.
Inoue K, Fry EA. Aberrant splicing of the DMP1-ARF/INK4a-MDM2-p53 pathway in cancer. Int J Cancer 2006;139:33-41.
Kamijo T, Zindy F, Roussel MF, Quelle DE, Downing JR, Ashmun RA, et al.
Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 1997;91:649-59.
Krimpenfort P, Quon KC, Mooi WJ, Loonstra A, Berns A. Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature 2001;413:83-6.
Sharpless NE, Bardeesy N, Lee KH, Carrasco D, Castrillon DH, Aguirre AJ, et al.
Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 2001;413:86-91.
Sherr CJ, Bertwistle D, DEN Besten W, Kuo ML, Sugimoto M, Tago K, et al.
P53-dependent and -independent functions of the arf tumor suppressor. Cold Spring Harb Symp Quant Biol 2005;70:129-37.
Stott FJ, Bates S, James MC, McConnell BB, Starborg M, Brookes S, et al.
The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2. EMBO J 1998;17:5001-14.
Itahana K, Zhang Y. Mitochondrial p32 is a critical mediator of ARF-induced apoptosis. Cancer Cell 2008;13:542-53.
Chen D, Tavana O, Chu B, Erber L, Chen Y, Baer R, et al.
NRF2 is a major target of ARF in p53-independent tumor suppression. Mol Cell 2017;68:224-32.e4.
Kim WY, Sharpless NE. The regulation of INK4/ARF in cancer and aging. Cell 2006;127:265-75.
He S, Sharpless NE. Senescence in health and disease. Cell 2017;169:1000-11.
Inoue K, Fry EA. Expression of p16INK4a
in human cancer – A new biomarker? Cancer Rep Rev 2018;2:1-7.
Zindy F, Quelle DE, Roussel MF, Sherr CJ. Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene 1997;15:203-11.
Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, et al.
Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 2006;443:421-6.
Molofsky AV, Slutsky SG, Joseph NM, He S, Pardal R, Krishnamurthy J, et al.
Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing. Nature 2006;443:448-52.
Kuo ML, den Besten W, Bertwistle D, Roussel MF, Sherr CJ. N-terminal polyubiquitination and degradation of the arf tumor suppressor. Genes Dev 2004;18:1862-74.
Inoue K, Roussel MF, Sherr CJ. Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1. Proc Natl Acad Sci U S A 1999;96:3993-8.
Parisi T, Pollice A, Di Cristofano A, Calabrò V, La Mantia G. Transcriptional regulation of the human tumor suppressor p14(ARF) by E2F1, E2F2, E2F3, and sp1-like factors. Biochem Biophys Res Commun 2002;291:1138-45.
Robertson KD, Jones PA. The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53. Mol Cell Biol 1998;18:6457-73.
Leone G, Nuckolls F, Ishida S, Adams M, Sears R, Jakoi L, et al.
Identification of a novel E2F3 product suggests a mechanism for determining specificity of repression by Rb proteins. Mol Cell Biol 2000;20:3626-32.
Aslanian A, Iaquinta PJ, Verona R, Lees JA. Repression of the Arf tumor suppressor by E2F3 is required for normal cell cycle kinetics. Genes Dev 2004;18:1413-22.
Zindy F, Eischen CM, Randle DH, Kamijo T, Cleveland JL, Sherr CJ, et al.
Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev 1998;12:2424-33.
Bouchard C, Lee S, Paulus-Hock V, Loddenkemper C, Eilers M, Schmitt CA, et al.
FoxO transcription factors suppress Myc-driven lymphomagenesis via direct activation of Arf. Genes Dev 2007;21:2775-87.
Qi Y, Gregory MA, Li Z, Brousal JP, West K, Hann SR, et al.
P19ARF directly and differentially controls the functions of c-Myc independently of p53. Nature 2004;431:712-7.
Krimpenfort P, Ijpenberg A, Song JY, van der Valk M, Nawijn M, Zevenhoven J, et al.
P15Ink4b is a critical tumour suppressor in the absence of p16Ink4a. Nature 2007;448:943-6.
Zhu S, Mott RT, Fry EA, Taneja P, Kulik G, Sui G, et al.
Cooperation between Dmp1 loss and cyclin D1 overexpression in breast cancer. Am J Pathol 2013;183:1339-50.
Kobayashi T, Wang J, Al-Ahmadie H, Abate-Shen C. ARF regulates the stability of p16 protein via REGγ-dependent proteasome degradation. Mol Cancer Res 2013;11:828-33.
Chen D, Shan J, Zhu WG, Qin J, Gu W. Transcription-independent ARF regulation in oncogenic stress-mediated p53 responses. Nature 2010;464:624-7.
Chio II, Sasaki M, Ghazarian D, Moreno J, Done S, Ueda T, et al.
TRADD contributes to tumour suppression by regulating ULF-dependent p19Arf ubiquitylation. Nat Cell Biol 2012;14:625-33.
Ko A, Shin JY, Seo J, Lee KD, Lee EW, Lee MS, et al.
Acceleration of gastric tumorigenesis through MKRN1-mediated posttranslational regulation of p14ARF. J Natl Cancer Inst 2012;104:1660-72.
Ko A, Han SY, Song J. Dynamics of ARF regulation that control senescence and cancer. BMB Rep 2016;49:598-606.
Wang X, Zha M, Zhao X, Jiang P, Du W, Tam AY, et al.
Siva1 inhibits p53 function by acting as an ARF E3 ubiquitin ligase. Nat Commun 2013;4:1551.
Boone DN, Qi Y, Li Z, Hann SR. Egr1 mediates p53-independent c-Myc-induced apoptosis via a noncanonical ARF-dependent transcriptional mechanism. Proc Natl Acad Sci U S A 2011;108:632-7.
Inoue K, Sherr CJ. Gene expression and cell cycle arrest mediated by transcription factor DMP1 is antagonized by D-type cyclins through a cyclin-dependent-kinase-independent mechanism. Mol Cell Biol 1998;18:1590-600.
Inoue K, Wen R, Rehg JE, Adachi M, Cleveland JL, Roussel MF, et al.
Disruption of the ARF transcriptional activator DMP1 facilitates cell immortalization, Ras transformation, and tumorigenesis. Genes Dev 2000;14:1797-809.
Inoue K, Zindy F, Randle DH, Rehg JE, Sherr CJ. Dmp1 is haplo-insufficient for tumor suppression and modifies the frequencies of Arf and p53 mutations in Myc-induced lymphomas. Genes Dev 2001;15:2934-9.
Sreeramaneni R, Chaudhry A, McMahon M, Sherr CJ, Inoue K. Ras-Raf-Arf signaling critically depends on the Dmp1 transcription factor. Mol Cell Biol 2005;25:220-32.
Mallakin A, Taneja P, Matise LA, Willingham MC, Inoue K. Expression of Dmp1 in specific differentiated, nonproliferating cells and its regulation by E2Fs. Oncogene 2006;25:7703-13.
Taneja P, Mallakin A, Matise LA, Frazier DP, Choudhary M, Inoue K, et al.
Repression of Dmp1 and Arf transcription by anthracyclins: Critical roles of the NF-kappaB subunit p65. Oncogene 2007;26:7457-66.
Mallakin A, Sugiyama T, Taneja P, Matise LA, Frazier DP, Choudhary M, et al.
Mutually exclusive inactivation of DMP1 and ARF/p53 in lung cancer. Cancer Cell 2007;12:381-94.
Mallakin A, Sugiyama T, Kai F, Taneja P, Kendig RD, Frazier DP, et al.
The Arf-inducing transcription factor Dmp1 encodes a transcriptional activator of amphiregulin, thrombospondin-1, JunB and Egr1. Int J Cancer 2010;126:1403-16.
Taneja P, Maglic D, Kai F, Sugiyama T, Kendig RD, Frazier DP, et al.
Critical roles of DMP1 in human epidermal growth factor receptor 2/neu-Arf-p53 signaling and breast cancer development. Cancer Res 2010;70:9084-94.
Fry EA, Taneja P, Maglic D, Zhu S, Sui G, Inoue K, et al.
Dmp1α inhibits HER2/neu-induced mammary tumorigenesis. PLoS One 2013;8:e77870.
Frazier DP, Kendig RD, Kai F, Maglic D, Sugiyama T, Morgan RL, et al.
Dmp1 physically interacts with p53 and positively regulates p53's stability, nuclear localization, and function. Cancer Res 2012;72:1740-50.
Maglic D, Zhu S, Fry EA, Taneja P, Kai F, Kendig RD, et al.
Prognostic value of the hDMP1-ARF-Hdm2-p53 pathway in breast cancer. Oncogene 2013;32:4120-9.
Maglic D, Stovall DB, Cline JM, Fry EA, Mallakin A, Taneja P, et al.
DMP1β, a splice isoform of the tumour suppressor DMP1 locus, induces proliferation and progression of breast cancer. J Pathol 2015;236:90-102.
Kendig RD, Kai F, Fry EA, Inoue K. Stabilization of the p53-DNA complex by the nuclear protein dmp1α. Cancer Invest 2017;35:301-12.
Inoue K, Mallakin A, Frazier DP. Dmp1 and tumor suppression. Oncogene 2007;26:4329-35.
Inoue K, Fry EA, Frazier DP. Transcription factors that interact with p53 and Mdm2. Int J Cancer 2016;138:1577-85.
Fry EA, Taneja P, Inoue K. Oncogenic and tumor-suppressive mouse models for breast cancer engaging HER2/neu. Int J Cancer 2017;140:495-503.
Inoue K, Fry EA. Haploinsufficient tumor suppressor genes. Adv Med Biol 2017;118:83-122.
Tschan MP, Federzoni EA, Haimovici A, Britschgi C, Moser BA, Jin J, et al.
Human DMTF1β antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation. Biochim Biophys Acta 2015;1849:1198-208.
Zheng Y, Zhao YD, Gibbons M, Abramova T, Chu PY, Ash JD, et al.
Tgfbeta signaling directly induces Arf promoter remodeling by a mechanism involving Smads 2/3 and p38 MAPK. J Biol Chem 2010;285:35654-64.
Zheng Y, Devitt C, Liu J, Iqbal N, Skapek SX. Arf induction by Tgfβ is influenced by sp1 and C/ebpβ in opposing directions. PLoS One 2013;8:e70371.
Linggi B, Müller-Tidow C, van de Locht L, Hu M, Nip J, Serve H, et al.
The t(8;21) fusion protein, AML1 ETO, specifically represses the transcription of the p14(ARF) tumor suppressor in acute myeloid leukemia. Nat Med 2002;8:743-50.
Shikami M, Miwa H, Nishii K, Kyo T, Tanaka I, Shiku H, et al.
Low p53 expression of acute myelocytic leukemia cells with t(8;21) chromosome abnormality: Association with low p14(ARF) expression. Leuk Res 2006;30:379-83.
Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M. The oncogene and polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 1999;397:164-8.
Aguilo F, Zhou MM, Walsh MJ. Long noncoding RNA, polycomb, and the ghosts haunting INK4b-ARF-INK4a expression. Cancer Res 2011;71:5365-9.
Jacobs JJ, Keblusek P, Robanus-Maandag E, Kristel P, Lingbeek M, Nederlof PM, et al.
Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers. Nat Genet 2000;26:291-9.
Cakouros D, Isenmann S, Cooper L, Zannettino A, Anderson P, Glackin C, et al.
Twist-1 induces Ezh2 recruitment regulating histone methylation along the Ink4A/Arf locus in mesenchymal stem cells. Mol Cell Biol 2012;32:1433-41.
Maglic D, Taneja P, Kendig RD, Kai F, Fry EA, Inoue K. Role of MEKK1 in Dmp1-Arf signaling. The 104th American Association of Cancer Research Annual Meeting. April, 2011. Cancer Res Suppl 2011;71:3076.
Maglic D, Kendig RD, Fry EA, Zhu S, Inoue K. MEKK1 regulates DMP1 transcriptional activity via phosphorylation and predicts breast cancer patient outcome. The 105th
American Association of Cancer Research Annual Meeting, April, 2012. Cancer Res Suppl 2012;72:4158.
Pasmant E, Laurendeau I, Héron D, Vidaud M, Vidaud D, Bièche I, et al.
Characterization of a germ-line deletion, including the entire INK4/ARF locus, in a melanoma-neural system tumor family: Identification of ANRIL, an antisense noncoding RNA whose expression coclusters with ARF. Cancer Res 2007;67:3963-9.
Popov N, Gil J. Epigenetic regulation of the INK4b-ARF-INK4a locus: In sickness and in health. Epigenetics 2010;5:685-90.
Kamijo T, Bodner S, van de Kamp E, Randle DH, Sherr CJ. Tumor spectrum in ARF-deficient mice. Cancer Res 1999;59:2217-22.
Chen D, Kon N, Li M, Zhang W, Qin J, Gu W, et al.
ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor. Cell 2005;121:1071-83.
Lee S, Cho YE, Kim SH, Kim YJ, Park JH. GLTSCR2 promotes the nucleoplasmic translocation and subsequent degradation of nucleolar ARF. Oncotarget 2017;8:16293-302.
Li Z, Hann SR. The myc-nucleophosmin-ARF network: A complex web unveiled. Cell Cycle 2009;8:2703-7.
Chen D, Yoon JB, Gu W. Reactivating the ARF-p53 axis in AML cells by targeting ULF. Cell Cycle 2010;9:2946-51.
Ruas M, Peters G. The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1998;1378:F115-77.
Gil J, Peters G. Regulation of the INK4b-ARF-INK4a tumour suppressor locus: All for one or one for all. Nat Rev Mol Cell Biol 2006;7:667-77.
Zhang Y, Xiong Y. Mutations in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53. Mol Cell 1999;3:579-91.
Lohrum MA, Ashcroft M, Kubbutat MH, Vousden KH. Contribution of two independent MDM2-binding domains in p14(ARF) to p53 stabilization. Curr Biol 2000;10:539-42.
Saporita AJ, Maggi LB Jr., Apicelli AJ, Weber JD. Therapeutic targets in the ARF tumor suppressor pathway. Curr Med Chem 2007;14:1815-27.
Nagy E, Beck Z, Kiss A, Csoma E, Telek B, Kónya J, et al.
Frequent methylation of p16INK4A and p14ARF genes implicated in the evolution of chronic myeloid leukaemia from its chronic to accelerated phase. Eur J Cancer 2003;39:2298-305.
Randerson-Moor JA, Harland M, Williams S, Cuthbert-Heavens D, Sheridan E, Aveyard J, et al.
Agermline deletion of p14(ARF) but not CDKN2A in a melanoma-neural system tumour syndrome family. Hum Mol Genet 2001;10:55-62.
Rizos H, Puig S, Badenas C, Malvehy J, Darmanian AP, Jiménez L, et al.
Amelanoma-associated Germline mutation in exon 1beta inactivates p14ARF. Oncogene 2001;20:5543-7.
Rutter JL, Goldstein AM, Dávila MR, Tucker MA, Struewing JP. CDKN2A point mutations D153spl (c. 457G & T) and IVS2+ 1G & T result in aberrant splice products affecting both p16INK4a and p14ARF. Oncogene 2003;22:4444-8.
Sargen MR, Merrill SL, Chu EY, Nathanson KL. CDKN2A mutations with p14 loss predisposing to multiple nerve sheath tumours, melanoma, dysplastic naevi and internal malignancies: A case series and review of the literature. Br J Dermatol 2016;175:785-9.
Prowse AH, Schultz DC, Guo S, Vanderveer L, Dangel J, Bove B, et al.
Identification of a splice acceptor site mutation in p16INK4A/p14ARF within a breast cancer, melanoma, neurofibroma prone kindred. J Med Genet 2003;40:e102.
Harland M, Taylor CF, Chambers PA, Kukalizch K, Randerson-Moor JA, Gruis NA, et al.
Amutation hotspot at the p14ARF splice site. Oncogene 2005;24:4604-8.
Williams RT, Barnhill LM, Kuo HH, Lin WD, Batova A, Yu AL, et al.
Chimeras of p14ARF and p16: Functional hybrids with the ability to arrest growth. PLoS One 2014;9:e88219.
Hewitt C, Lee Wu C, Evans G, Howell A, Elles RG, Jordan R, et al.
Germline mutation of ARF in a melanoma kindred. Hum Mol Genet 2002;11:1273-9.
Silva J, Domínguez G, Silva JM, García JM, Gallego I, Corbacho C, et al.
Analysis of genetic and epigenetic processes that influence p14ARF expression in breast cancer. Oncogene 2001;20:4586-90.
Silva J, Silva JM, Domínguez G, García JM, Cantos B, Rodríguez R, et al.
Concomitant expression of p16INK4a and p14ARF in primary breast cancer and analysis of inactivation mechanisms. J Pathol 2003;199:289-97.
Song X, Sturgis EM, Huang Z, Li X, Li C, Wei Q, et al.
Potentially functional variants of p14ARF are associated with HPV-positive oropharyngeal cancer patients and survival after definitive chemoradiotherapy. Carcinogenesis 2014;35:62-8.
Pietersen AM, Horlings HM, Hauptmann M, Langerød A, Ajouaou A, Cornelissen-Steijger P, et al.
EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer. Breast Cancer Res 2008;10:R109.
Parrales A, Iwakuma T. Targeting oncogenic mutant p53 for cancer therapy. Front Oncol 2015;5:288.
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