Cellular Senescence Promotes Skin Carcinogenesis through p38MAPK and p44/42MAPK Signaling

Abstract

Cellular senescence entails an irreversible growth arrest that evolved in part to prevent cancer. Paradoxically, senescent cells secrete proinflammatory and growth-stimulatory molecules, termed the senescence-associated secretory phenotype (SASP), which is correlated with cancer cell proliferation in culture and xenograft models. However, at what tumor stage and how senescence and the SASP act on endogenous tumor growth in vivo is unknown. To understand the role of senescence in cancer etiology, we subjected p16-3MR transgenic mice, which permit the identification and selective elimination of senescent cells in vivo, to the well-established two-step protocol of squamous cell skin carcinoma, in which tumorigenesis is initiated by a carcinogen 7,12-dimethylbenz[α]anthracene, and then promoted by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that TPA promotes skin carcinogenesis by inducing senescence and a SASP. Systemic induction of senescence in nontumor-bearing p16-3MR mice using a chemotherapy followed by the two-step carcinogenesis protocol potentiated the conversion of benign papillomas to carcinomas by elevating p38MAPK and MAPK/ERK signaling. Ablation of senescent cells reduced p38MAPK and MAPK/ERK signaling, thereby preventing the progression of benign papillomas to carcinomas. Thus, we show for the first time that senescent cells are tumor promoters, not tumor initiators, and that they stimulate skin carcinogenesis by elevating p38MAPK and MAPK/ERK signaling. These findings pave the way for developing novel therapeutics against senescence-fueled cancers. SIGNIFICANCE: These findings identify chemotherapy-induced senescence as a culprit behind tumor promotion, suggesting that elimination of senescent cells after chemotherapy may reduce occurrence of second cancers decades later. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3606/F1.large.jpg.

Figure 1:. TPA induces senescence and a SASP in human keratinocytes and mouse skin

(A-C) Human keratinocytes were treated with DMSO (control) or TPA (10 nM) for 48 h. 12 d later, the cells were stained for SA-ß-gal (A-B) and assessed for cell proliferation (C). N=3 independent experiments. (D) Total RNA was isolated from human keratinocytes and analyzed for p16, p21 and SASP mRNAs normalized to actin. N=3 independent experiments, presented as means ±SD; **p<0.01, ***p<0.001, ****p<0.0001. (E) Protein lysates from human keratinocytes treated with DMSO or TPA (5 nM or 10 nM) were evaluated for Lmnb1 and p16 levels by immunoblotting. (F) Total RNA was isolated from mouse skin topically treated with either acetone or TPA for 2 mos. One month after the last treatments, the skin was analyzed for the indicated mRNAs, normalized to actin and tubulin. N=5 for the acetone group, N=7 for the TPA group. Shown are means ±SEM; *p<0.05, **p<0.01 (Student t-test). (G-H) Frozen tissue sections of acetone- or TPA-treated skin were stained for SA-ß-gal (blue) and nuclei (red). (G) Representative images are shown (4x magnification), and arrows denote SA-ß-gal positive areas. (H) Percent of SA-ß-gal positive surface area of skin compared to unstained skin. N=5 for acetone group, N=7 for TPA group. Shown are means ±SEM; *p<0.05 (Student t-test).

Figure 2:. TPA-induced senescence (pre-treatment) stimulates skin tumor growth

(A) Schematic of treated p16–3MR mice. (B) Representative images of tumor- bearing mice, 24 wks after DMBA treatment. (C) Tumors of Acetone + PBS, Acetone + GCV, TPA + PBS, TPA + GCV groups were monitored once a wk; mean tumor volume over 24 wks is shown. Shown are means ±SEM; **p<0.01, ***p<0.001, ****p<0.0001 (two-way ANOVA, Tukey’s test for multiple comparisons was used post-analyses). (D) Luminescence of tumor-bearing skin in arbitrary units (A.U.) units 24 wks after DMBA treatment. N = 8–10 per group. Shown are means ±SEM; *p<0.05 (one-way ANOVA, Sidak’s multiple comparisons test was used post-analyses). (E) Representative images of H&E staining of skin (top panels) and tumors (middle panels) (200 μm and 50 μm respectively), and Ki-67 staining of tumors (bottom panels) (50 μm). (F) Percentage of Ki-67-positive cells in tumors from the 4 treatment groups. Shown are means ±SEM; *p<0.05 (one-way ANOVA, Sidak’s multiple comparisons test was used post-analyses).

Figure 3:. DOXO induces senescence and a SASP in human keratinocytes and mouse skin

(A-C) Human keratinocytes were treated with DMSO or DOXO (250 nM) for 24 h. 10 d later, the cells were stained for SA-ß-gal (A-B) and assessed for cell proliferation (C). N=3 independent experiments. Shown are means ±SD, *p<0.5, ***p<0.001, (Student t test). (D) Total RNA was isolated from the indicated cells and analyzed for p16, p21 and SASP mRNAs, normalized to actin. N=3 independent experiments. Shown are means ±SD; **p<0.01, ***p<0.001. (E) Protein lysates from human keratinocytes were evaluated for p16 and LMNB1 by immunoblotting. (F) Total RNA was isolated from the skin of PBS- or DOXO-treated mice and analyzed for the indicated mRNAs, normalized to actin and tubulin. N=6 per each treatment group. Shown are means ±SEM; *p<0.05, **p<0.01 (Student t-test). (G-H) Representative image (G) and quantification in arbitrary units (A.U.) of whole body luminescence of p16–3MR mice one mo after PBS or DOXO treatments. N=6 per each treatment group. Shown are means ±SEM; *p<0.05 (Student t test).

Figure 4:. DOXO-induced senescence fuels skin tumor growth

(A) A schematic of the PBS/DOXO and skin carcinogenesis regimens of p16–3MR mice. TPA treatment lasted 16 wks. (B) Quantification of whole body luminescence of p16–3MR mice 10 d after PBS/DOXO treatments and one wk after DMBA initiation. Quantification is in arbitrary units (A.U.). Shown are means ±SEM; *p<0.05 (one-way ANOVA, Sidak’s multiple comparisons test was used post-analyses). (C) Total RNA was isolated from the skin of the 4 treatments groups and analyzed for p16 normalized to actin and tubulin (PBS + PBS: n=10, PBS + GCV: n=8, DOXO + PBS: n=9, DOXO + GCV: n=8). Shown are means ±SEM; *p<0.05, **p<0.01 (one-way ANOVA, Sidak’s multiple comparisons test was used post-analyses). (D) Representative images of tumor-bearing mice 21 wks after DMBA treatment and 5 wks after TPA treatment in PBS + PBS, PBS + GCV, DOXO + PBS, DOXO + GCV groups. (E) The mean tumor volume of the same mice is shown over 21 wks. Shown are means ±SEM; *p<0.05, **p<0.01, ****p<0.0001 (two way ANOVA, Tukey’s test for multiple comparisons was used post-analyses). (F-G) Frozen skin sections of (PBS + PBS: n=10, PBS + GCV: n=10, DOXO + PBS: n=14, DOXO + GCV: n=14) treatment groups were stained for SA-ß-gal (blue) and nuclei (red). (F) Representative images are shown (50 μm), and arrows denote SA-ß-gal positive areas. (G) Percent of SA-ß-gal positive surface area of skin compared to non-stained skin. Shown are means ±SEM; *p<0.5, **p<0.01, ***p<0. 001 (one-way ANOVA, Sidak’s multiple comparisons test was used post-analyses).

Figure 5:. Eliminating DOXO-induced senescent cells prevents malignant tumor development

Representative images of H&E staining of the skin (top panels) and tumors (bottom panels) in PBS + PBS, PBS + GCV, DOXO + PBS, DOXO + GCV treated-groups (200 μm). (B-D) In the same 4 groups (PBS + PBS: n =9, PBS + GCV: n=7, DOXO + PBS: n=11, DOXO + GCV: n=7), representative IHC images for Ki-67 (B), vimentin (C) and CD-31 (D) (50 μm) with the corresponding quantification of percent positive cells in the right panels. Shown are means ±SEM; *p<0.05, **p<0.01, ***p<0. 001 (one-way ANOVA, Sidak’s multiple comparisons test was used post-analyses).

Figure 6:. The SASP drives skin carcinogenesis

(A-D) Representative images and quantification of tumorspheres of A-431 cells embedded in reduced growth factor Matrigel incubated for 7 d with conditioned media from non-senescent (quiescent) or senescent HCA2 fibroblasts with or without SB203580 (A-B) or anti-IL1α (C-D). (A and C) Tumorspheres are shown stained with DAPI (blue) and EdU (green). (B and D) Quantification of percentage EdU-positive cells. N=3 independent experiments. Shown are mean ±SEM; **p<0.01, ****p<0.0001 (one-way ANOVA, Sidak’s multiple comparisons test was used post-analyses). (E) Representative tumor and adjacent skin luminescence images of PBS- or DOXO-treated mice, 21 wks after DMBA treatment and 5 wks after the last TPA treatment. Luminescent skin and tumors are labeled on the images as (S) and (T), respectively. (F) Luminescence quantification of tumors and adjacent skin shown in (E) in arbitrary units (A.U.). N= 8 per each treatment group. Shown are mean ±SEM; ***p<0.001 (Student t test). (G) Luciferase activity of untreated skin compared to DOXO-tumor, protein lysates were normalized to total protein content. N= 4–5 for skin and DOXO tumors respectively. Shown are mean ±SEM; *p<0.05 (Student t test). (H) Total RNA was isolated from skin adjacent to tumors from mice in (E-F) and analyzed for the indicated mRNAs, normalized to actin and tubulin. N= 8 per treatment group. Shown are mean ±SEM; *p<0.5, **p<0.001 (Student t test).

Figure 7:. Phospho-p38 and phospho-p44/42 MAPK signaling is increased in tumors from DOXO-treated mice

(A-B) Representative IHC images of tumors from PBS + PBS: n =9, PBS + GCV: n=7, DOXO + PBS: n=11, DOXO + GCV: n=7, stained with p-p38 (A) or p-p44/42 (B) with the corresponding quantification of percent positive cells in the right panels. Shown are means ±SEM; *p<0.05, **p<0.01, (one-way ANOVA, Sidak’s multiple comparisons test was used post-analyses). (C-D) Representative IHC images of normal human skin and hSCC lesions (grades I-III) stained with p-p38 (C) and p-p44/42 (D). (E) Working model showing that DOXO induces senescence and a SASP in keratinocytes and fibroblasts, creating a microenvironment permissive for tumor growth and invasion. Once tumors are formed, senescent cells within the tumors also reinforce tumor growth by elevating p-p38 and p-p44/42 signaling.