Overcoming cellular senescence in human cancer pathogenesis

Abstract

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and -3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P = 0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11-q12 (0.99) and +8p22-pter (0.94) in the immortal muscle invasive TCCs, and of -9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13-p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P = 0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P = 0.04) than was p16 or pRb alteration (P = 0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11-q12, -3p13-p14, or -8p21-pter.

Figure 1

Morphology of TCC compared to HUC in culture. (A) Normal HUCs show typical epithelial morphology with tightly adherent monolayers of polygonal cell. Note the lack of mesenchymal cell contamination. Original magnification, 200×. (B) Superficial papillary TCCs also show similar epithelial cellular morphology in vitro, but unlike normal HUCs, often form three dimensional structures at the leading edge of growth reminiscent of their in vivo multilayered morphology. Original magnification, 200×. (C) Myoinvasive papillary TCC 96-2 also formed a cell line that retained its ability to form three-dimensional structures after prolonged culture. Original magnification, 200×. (D) Myoinvasive TCC 96-1 with flat morphology in vitro is shown here. Original magnification, 200×.

Figure 2

Western blot analysis for p16 at P0 and at senescence in superficial TCCs. p16 was elevated in all of the six superficial TCCs at senescence compared to P0, similar to the result obtained with normal HUC. Note also that some of the superficial TCCs showed p16 in the P0 culture. These superficial TCCs senesced earlier in vitro than TCCs with undetectable p16 at P0.

Figure 3

Shown here is an example of staining for β-galactosidase activity at P0 and at senescence in the same superficial TCC sample TCC 96-12. (A) The proliferating TCC culture shows no β-galactosidase activity; (B) the same TCC culture, when senescent, shows strong β-galactosidase activity. Original magnification, 100×.

Figure 4

Western blot analysis at approximately P15 for p16 and pRb in the six myoinvasive TCC cell lines in this study. Three myoinvasive TCCs show no detectable p16, whereas three show no detectable pRb. Results done at P0 show the same results. The nonspecific band is shown to demonstrate protein loading.

Figure 5

Southern blot analysis for CDKN2/p16 methylation. Flanking cut (F.C.) HUC was digested only with the enzyme for the F.C. EcoRI. All other samples were digested with both EcoRI and SacII. TSU–PR1 is a prostate cell line containing methylated CDKN2/p16 that was used as a positive control for methylation. The three TCC cell lines that expressed elevated p16 showed no altered methylation. In contrast, the three TCCs that failed to show p16 by Western blot analysis showed genetic alterations by Southern blot analysis. TCC 96-1 and TCC 97-1 both contained homozygous CDKN2/p16 deletions. TCC 94-10 had one methylated allele. Sequencing of cDNA showed a mutation in the second allele as discussed in text.

Figure 6

Western blot analysis of myoinvasive TCCs for p53. Note relatively low, but detectable, p53 levels at P0 and in senescent HUCs and in one myoinvasive TCC. In contrast, note altered p53 levels in five myoinvasive TCCs, four showing stabilized p53 and one TCC lacking a detectable p53 signal. Notably, one of the TCC with stabilized p53, TCC 97-1, had wild-type TP53 by sequencing. Results of later passage cultures are shown, but results were the same at P0. Thus, no changes occurred in culture.

Figure 7

Chromosome arm losses and gains in myoinvasive TCCs as detected by CGH. (A,B) Chromograms recording the number of six myoinvasive TCCs exhibiting a change on each of 39 nonacrocentric chromosome arms. Horizontal hash marks indicate that the loss or gain on both arms was linked. Diagonal marks indicate that the loss or gain was not linked to the other chromosomal arms. (C,D) Probabilities calculated under the simple stochastic model conditional on the chromograms. The arm has an elevated rate of loss or gain. (A,C) Late0stage losses; (B,D) late-stage gains. Results of calculations are presented in the text.