Abstract
Human chromosome 9p21.3 is susceptible to inactivation in cell immortalization and diseases, such as cancer, coronary artery disease and type-2 diabetes. Although this locus encodes three cyclin-dependent kinase (CDK) inhibitors (p15INK4B, p14ARF and p16INK4A), our understanding of their functions and modes of action is limited to the latter two. Here, we show that in vitro p15INK4B is markedly stronger than p16INK4A in inhibiting pRb1 phosphorylation, E2F activity and cell-cycle progression. In mice, urothelial cells expressing oncogenic HRas and lacking p15INK4B, but not those expressing HRas and lacking p16INK4A, develop early-onset bladder tumors. The potency of CDKN2B/p15INK4B in tumor suppression relies on its strong binding via key N-terminal residues to and inhibition of CDK4/CDK6. p15INK4B also binds and inhibits enolase-1, a glycolytic enzyme upregulated in most cancer types. Our results highlight the dual inhibition of p15INK4B on cell proliferation, and unveil mechanisms whereby p15INK4B aberrations may underpin cancer and non-cancer conditions.
Highlights
Human chromosome 9p21.3 is susceptible to inactivation in cell immortalization and diseases, such as cancer, coronary artery disease and type-2 diabetes
Unlike p14ARF and p16INK4A whose tumorsuppressive activities have been well documented, p15INK4B is often regarded as functionally equivalent to p16INK4A and a bystander during 9p21.3 deletion. These notions are, in stark contrast to the phenomena that (i) p15INK4B can be targeted for deletion independently of p14ARF and p16INK4A2,12; (ii) the three cyclin-dependent kinase (CDK) inhibitors can be down-regulated separately by promoter-specific methylation, cis-acting long non-coding antisense RNA (i.e., ANRIL and c-ANRIL)[13,14,15] or divergent upstream signals (i.e., p15INK4B by transforming growth factor beta (TGF-beta)16,17); (iii) p15INK4B becomes critically important in the absence of p16INK4A 18; and (iv) in genetically engineered mice the loss of both p14ARF and p16INK4A fails to cooperate with oncogenes to elicit tumors in certain cell types, such as urothelium[19]
We crossed our Upk2-HRas transgenic mice, in which the expression of oncogenic HRas was driven by urothelium-restricted murine Upk[2] promoter (Fig. 1a)[19,27], with ubiquitous CDKN2B knockout mice, in which exon 2 of CDKN2B was replaced with a neo gene (Fig. 1a), which ablated p15 expression[28]
Summary
Human chromosome 9p21.3 is susceptible to inactivation in cell immortalization and diseases, such as cancer, coronary artery disease and type-2 diabetes. To understand the mechanisms underlying the different potencies of p15 and p16 in urothelial tumor suppression, we examined cell-cycle distribution by fluorescence-activated cell sorting (Fig. 3b) and pRB1 phosphorylation by western blotting (Fig. 3c) in the in vivo mouse cohort as shown, e and Supplementary Fig. 1.
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