Cellular senescence promotes skin carcinogenesis through p38MAPK and p44/p42MAPK signaling

The therapeutic induction of senescence is a potential means to treat cancer, primarily acting through the induction of a persistent growth‐arrested state in tumors. However, recent studies have indicated that therapy‐induced senescence (TIS) in tumor cells allows for the prolonged survival of a subgroup of cells in a dormant state, with the potential to re‐enter the cell cycle along with an increased stemness gene expression. Residual cells after TIS with increased cancer stem cell phenotype may have profound implications for tumor aggressiveness and disease recurrence. Herein, we investigated senescence‐associated stemness in EpCAM+/CD133+ liver cancer stem cell and EpCAM−/CD133− nonstem cell populations in HuH7 cell line. We demonstrated that treatment with doxorubicin induces senescence in both cell populations, accompanied by a significant increase in the expression of reprogramming genes SOX2, KLF4, and c‐MYC as well as liver stemness‐related genes EpCAM, CK19, and ANXA3 and the multidrug resistance‐related gene ABCG2. Moreover, doxorubicin treatment significantly increased EpCAM + population in nonstem cells indicating senescence‐associated reprogramming of nonstem cell population. Also, Wnt/β‐catenin target genes were increased in these cells, while inhibition of this signaling pathway decreased stem cell gene expression. Importantly, Dox‐treated EpCAM−/CD133− nonstem cells had increased in vivo tumor‐forming ability. In addition, when SASP‐CM from Dox‐treated cells were applied onto hİPSC‐derived hepatocytes, senescence was induced in hepatocytes along with an increased expression of TGF‐β, KLF4, and AXIN2. Importantly, SASP‐CM was not able to induce senescence in Hep3B‐TR cells, a derivative line rendered resistant to TGF‐β signaling. Furthermore, ELISA experiments revealed that the SASP‐CM of Dox‐treated cells contain inflammatory cytokines IL8 and IP10. In summary, our findings further emphasize the importance of carefully dissecting the beneficial and detrimental aspects of prosenescence therapy in HCC and support the potential use of senolytic drugs in HCC treatment in order to eliminate adverse effects of TIS.

Squamous cell carcinoma (SCC) is the second most common non-melanoma skin cancer, and has a high rate of recurrence and metastasis. Systemic genotoxic chemotherapeutic drugs such as doxorubicin (Doxo) can be used to treat SCC, but these agents can have deleterious long-term side effects, including fueling the development of more aggressive cancers. Some of the adverse effects of genotoxic chemotherapies might be due to their ability to induce cellular senescence. Cellular senescence is a tumor suppressive mechanism that entails a permanent cell growth arrest accompanied by several metabolic changes. Senescent cells increase during aging, and accumulating evidence suggests they can contribute to a variety of age related pathologies, including cancer, by secreting numerous pro-inflammatory molecules, growth factors and proteases, a feature termed the senescence-associated secretory phenotype (SASP). We recently showed that genotoxic and cytotoxic chemotherapeutic agents induce senescence and a SASP in vivo. However, a direct role for senescent cells in skin carcinogenesis in vivo remains unexplored. To investigate the role of senescent cells and the SASP in squamous cell skin carcinogenesis, we first induced senescence with Doxo in p16-3MR transgenic mice. These mice are designed to permit the selective elimination of senescent cells expressing the established senescence marker p16INK4a (p16) by administrating ganciclovir (GCV) which has a high affinity for the herpes simplex viral thymidine kinase (HSV-TK). Phosphorylated GCV is a DNA chain terminator that kills HSV-TK expressing cells by apoptosis. After Doxo treatment, we initiated skin SCC in p16-3MR mice with 7, 12-dimethylbenz[a]anthracene (DMBA), followed by treatment with 12-O-Tetradecanoylphorbol-13-acetate (TPA) to promote skin carcinogenesis. 25 weeks after Doxo treatment, we observed a significant increase in p16 mRNA levels as well as an increase in expression of the SASP factors IL-1α, MMP-9, and IL-17 in the skin of Doxo- compared to PBS- treated mice. Further, the presence of senescent cells resulted in significantly larger and more proliferative tumors compared to PBS-treated mice. Importantly, systemic elimination of Doxo-induced senescent cells using GCV reduced the expression of SASP factors and significantly reduced tumor size, suggesting that senescent cells fuel skin tumor progression. These findings will help our understanding of the role of chemotherapy-induced cellular senescence in skin carcinogenesis and will pave the way for developing novel therapeutics against squamous cell carcinoma and the deleterious side effects of genotoxic chemotherapies. Citation Format: Fatouma Alimirah, Alexis Valdovinos, Emily Chang, Sena Alptekin, Tanya Pulido, Elijah Jones, Chandani Limbad, Michael Velarde, Marco Demaria, Albert Davalos, Judith Campisi. Cellular senescence drives skin carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 466.

<div>Abstract<p>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 <i>in vivo</i> 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 <i>in vivo</i>, 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.</p>Significance:<p>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.</p></div>