Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype

Abstract

Cellular senescence suppresses cancer by preventing the proliferation of cells that experience potentially oncogenic stimuli. Senescent cells often express p16(INK4a), a cyclin-dependent kinase inhibitor, tumor suppressor, and biomarker of aging, which renders the senescence growth arrest irreversible. Senescent cells also acquire a complex phenotype that includes the secretion of many cytokines, growth factors, and proteases, termed a senescence-associated secretory phenotype (SASP). The SASP is proposed to underlie age-related pathologies, including, ironically, late life cancer. Here, we show that ectopic expression of p16(INK4a) and another cyclin-dependent kinase inhibitor, p21(CIP1/WAF1), induces senescence without a SASP, even though they induced other features of senescence, including a stable growth arrest. Additionally, human fibroblasts induced to senesce by ionizing radiation or oncogenic RAS developed a SASP regardless of whether they expressed p16(INK4a). Cells induced to senesce by ectopic p16(INK4a) expression lacked paracrine activity on epithelial cells, consistent with the absence of a functional SASP. Nonetheless, expression of p16(INK4a) by cells undergoing replicative senescence limited the accumulation of DNA damage and premature cytokine secretion, suggesting an indirect role for p16(INK4a) in suppressing the SASP. These findings suggest that p16(INK4a)-positive cells may not always harbor a SASP in vivo and, furthermore, that the SASP is not a consequence of p16(INK4a) activation or senescence per se, but rather is a damage response that is separable from the growth arrest.

FIGURE 1.

p16^INK4a and p21^CIP1/WAF1 induce a distinct secretory profile. A, ELISA measurements of IL-6, IL-8, and GROα. We analyzed CM from the indicated presenescent (PRE) cell strains and cells induced to senesce (SEN) by p16^INK4a (p16) or p21^CIP1/WAF1 (p21) overexpression, irradiation (XRA), replicative exhaustion (REP), or oncogenic RAS^V12 (RAS). CM from untreated and control-infected PRE cells were pooled. The asterisk denotes a value higher than the limit of the scale (see supplemental Fig. 1B for additional ELISA data, including GROα). B, mRNA profiles of p16^INK4a- and p21^CIP1/WAF1-induced senescent cells. TaqMan PCR was used to quantify the indicated mRNAs in PRE WI-38 and IMR-90 cells (baseline, n = 15; WI-38 = 9 and IMR90 = 6), PRE cells infected with an insertless lentivirus (PRE L3P, n = 5; WI-38 = 3, and IMR90 = 2), cells made senescent by infecting with lentiviruses that overexpress p16^INK4a (p16, n = 5; WI-38 = 3 and IMR90 = 2) or p21^CIP1/WAF1 (p21, n = 2; IMR90 = 2), and x-ray-induced senescent cells (XRA, n = 8; WI-38 = 5 and IMR90 = 3) (see also supplemental Fig. 1C). C, secretory profile of p16^INK4a-induced senescent cells. CM from cells induced to senesce by p16^INK4a overexpression (p16) were analyzed by antibody arrays and compared with replicatively or x-ray-induced senescent cells (REP; XRA). Signals from WI-38 and IMR-90 cells were averaged, and the PRE secretory profile was used as baseline. Log₂ fold variations from the baseline are color-coded (yellow, signals higher than baseline; blue, signals below baseline). The 41 factors previously identified as significantly changed between PRE and SEN cells are shown (12). The number of samples analyzed is shown on the right. D, correlation between the secretory profiles of REP and p16^INK4a-induced senescent (p16) cells. The trend line corresponds to the linear regression comparing the two populations (correlation = 0.22, slope = 0.12). E, unsupervised hierarchical clustering analysis of secretory profiles of PRE and SEN WI-38 cells, using all 120 factors interrogated by the antibody arrays. SEN(p16) cells cluster away from SEN(XRA), SEN(REP) or SEN(RAS) cells (red arrow and broken line).

FIGURE 2.

p16^INK4a status does not influence the secretory profile of senescent cells. A, comparison of the most highly secreted cytokines of control (L3P>REP) WI-38 cells, which express high levels of p16^INK4a at replicative senescence (REP), and REP WI-38 cells depleted of p16^INK4a owing to shp16 expression (shp16>REP). The graph (right) shows the overall correlation between 41 SASP factors in both populations (trend line = linear regression; correlation = 0.72, slope = 0.79). B, unsupervised hierarchical clustering analysis of PRE and SEN WI-38 cells using 120 factors interrogated by the antibody arrays. Cells induced to senesce by irradiation (XRA), replicative exhaustion (REP), and oncogenic RAS^V12 (RAS) cluster with p16-negative SEN(REP) cells (shp16>REP) (red arrow and broken line). C, quantitative measurements of IL-6 and IL-8 secretion. CM from PRE and SEN(REP) control (L3P>REP) and p16^INK4a-depleted (shp16>REP) WI-38 cells were analyzed by ELISA. D, immunofluorescence detection of p16^INK4a, IL-6, and IL-8 in SEN (shp16>REP) WI-38 cells. Each panel is a different field. E, mRNA profiles of cells induced to senesce with or without p16^INK4a expression. TaqMan PCR was used to quantify the indicated mRNAs in PRE WI-38 cells (baseline), control SEN(REP) cells (L3P>REP), and p16INK4a-depleted SEN(REP) cells (shp16>REP) cells.

FIGURE 3.

Establishment and maintenance of the SASP are p16^INK4a-independent. A, coimmunostaining for p16^INK4a and IL-6 in SEN(XRA), SEN(REP), SEN(RAS) WI-38 cells showing that single p16^INK4a-expressing cells can also express high levels of IL-6. B, effect of p16^INK4a-depletion on IL-6 secretion by senescent cells. WI-38 cells were infected with control (insertless), p16^INK4a or shp16-expressing lentiviruses and then induced to senesce by irradiation (XRA) or oncogenic RAS (RAS). CM were analyzed for IL-6 by ELISA. IL-6 secretion by control-infected SEN(XRA) was used as the baseline (arbitrary value = 1). p values were determined by Student's t test.

FIGURE 4.

p16^INK4a-depleted PRE cells develop early IL-6 secretion with 53BP1 foci formation. A, IL-6 secretion as a function of passage. CM from WI-38 cells infected with control (insertless) or shp16 lentivirus were analyzed for IL-6 by ELISA. We compared early (early PRE) and late (late PRE) passage cells with the same cells at complete replicative senescence (SEN(REP)). B, single cell analysis of p16^INK4a and 53BP1 foci. p16^INK4a and 53BP1 foci were detected by immunofluorescence. Upper panels show p16^INK4a (green) and 53BP1 (red) co-staining. Middle and bottom panels show the grayscale for 53BP1 staining. The yellow arrows indicate cells with three or more 53BP1 foci. C, cell populations shown in B were analyzed for the percentage of nuclei positive for three or more 53BP1 foci. At least 200 nuclei were scored per condition.

FIGURE 5.

p16^INK4a-induced senescent human and mouse fibroblasts do not alter epithelial cell phenotypes. A, cells induced to senesce by p16^INK4a overexpression do not promote epithelial scattering. ZR75.1 human breast epithelial cells were incubated with CM from PRE WI-38 cells or cells induced to senesce by p16INK4a overexpression (SEN(p16) or irradiation (SEN(XRA)). B, epithelial cell scattering depends on secreted IL-6 and IL-8. CM from PRE, SEN(XRA) and SEN(p16) WI-38 cells were incubated with ZR75.1 epithelial cells as in A. CM were supplemented with recombinant IL-6 and IL-8, IL-6 and IL-8 blocking antibodies, or control IgG. Clumps sizes were quantified using a Cellomix analyzer. C, cells induced to senesce by p16^INK4a do not stimulate epithelial cell proliferation. SCp2 mouse mammary epithelial cells were incubated with CM from PRE or SEN cells. Senescence was induced by p16^INK4a overexpression (p16), irradiation (XRA), or replicative exhaustion (REP). Epithelial cell number was determined after 7 days and is expressed relative the value obtained with PRE CM. D, mouse fibroblasts induced to senesce by p16^INK4a do not stimulate epithelial cell proliferation. SCp2 cells were incubated with CM from PRE or SEN primary mouse embryo fibroblasts or 10% serum. Senescence was induced by p16^INK4a overexpression (p16) or irradiation (XRA). Epithelial cell number was determined as described in C. Student's t test, ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; #, p > 0.1.