Aberrant Expression of p14ARF in Human Cancers: A New Biomarker?

Abstract

The ARF and INK4a genes are located on the CDKN2a locus, both showing tumor suppressive activity. ARF has been shown to monitor potentially harmful oncogenic signalings, making early stage cancer cells undergo senescence or programmed cell death to prevent cancer. Conversely, INK4a detects both aging and incipient cancer cell signals, and thus these two gene functions are different. 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, frame shift, 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 pre-malignant 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; BMI1; CDKN2a; DMP1 (DMTF1); E2F; INK4a; biomarker; cancer; expression; p53; prognosis.

Figure 1.

The structure for the human p15^INK4b-p14^ARF-p16^INK4a locus. 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 1β 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 p15^INK4b in humans (from 3’ of exon 2 for p15^INK4b to 5’ of exon 1β). All of p15^Ink4b, p19^Arf, and p16^Ink4a genes act as tumor suppressors as reported by Krimpenfort et al. (8, 90). 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 (40). Pasmant et al. identified a new large antisense noncoding RNA (named ANRIL; 59, 60) to this genomic locus, with a first exon located in the promoter of the p14^ARF gene and overlapping the two exons for p15^CDKN2b. Expression of ANRIL was simultaneously found with p14^ARF both in physiologic and pathologic conditions. Kobayashi et al. found that that p14^ARF regulates the stability of the p16^INK4a protein in human and mouse cells (91). Importantly, ARF promoted rapid degradation of p16^INK4a 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 (91). ULF, MKRN1, and Siva1 are E3 ligases for ARF that accelerates its degradation (, , –70).

Figure 2.. Oncogenic and tumor suppressive signaling involving Dmp1 and Arf.

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 black. The output signals for Arf, Mdm2, and p53 are shown in striped arrows. 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 arrows. Conversely, negative signals for Arf have been shown in black. The output signals for Arf, Mdm2, and p53 are shown in striped arrows. Dmp1α transactivates the Arf promoter in response to oncogenic stresses, and physically interacts with p53 to neutralize all activities of Mdm2 to activate the p53 pathway in response to DNA damage (–3). Both Dmp1^−/− and Dmp1^+/− mice show hypersensitivity to develop tumors in response to carcinogen or γ-irradiation (32, 33). 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 (31, 40). 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 (34, 37, 39). The human DMP1 locus generates three splice variants, namely DMP1α, β, and γ with antagonizing activity between DMP1α and β (46). DMP1β 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 (42). 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 (42); Dmp1 also stabilized p53 binding to transcriptional target genes (45). Dmp1α-p53 interaction increases the levels of p53 independent of Dmp1’s DNA-binding, and hence both p21^Cip1 and Bbc3 promoters were synergistically activated by co-expression of Dmp1α and p53 in p53^−/−; Arf^−/− cells (42). In accordance, the induction of p21^Cip1 and Bbc3 by genotoxic drug treatment was more seriously affected in Dmp1^−/− and p53^−/− tissues than in Arf^−/− (42). In summary, Dmp1α stimulates the p53 pathway by direct transactivation of the Arf promoter in response to oncogenic stresses (34, 37, 39, 40) and direct physical interaction with p53 in DNA damage response (DDR; 42, 45). 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. There are feed forward/back regulations between p53 and Mdm2, p53 and Arf, and p53 and Dmp1. In addition, Arf negatively regulates Mdm2, and Mdm2 negatively regulates p53 as published.