How Do Senescent Cells Fuel Certain Cancers?

Repurposing the FOXO4 senolytic against triple-negative breast cancer

“Here we show that an anti-senescence compound can target metastatic cancers. We found that, in TNBCs, mutant p53 attained a distinctive conformation that has novel oncogenic roles through binding to the transcription factor Forkhead box O (FOXO) 4 sequestered within promyelocytic leukemia (PML) foci.

Since these nuclear structures are specific to senescent cells, we tested the senolytic FOXO4 peptide. In cytotoxicity experiments, we found this compound to target TNBCs specifically over other breast cancer subtypes. Most importantly, these compounds decrease metastatic burden in the most commonly-used mouse model for human breast cancer metastasis. In summary, our results demonstrate that mutant p53-driven cancers presented senescent cell-specific characteristics that makes them a great candidate for the FOXO4-directed anti-senescence therapy. We expect that creative repurposing of senolytics to translate to other types of cancer that are driven by mutant p53.” (1)

Senotherapeutics in Cancer and HIV

“FOXO4 is a transcription factor that plays a role in the maintenance of senescent cell viability. In addition, its presence in a small fraction of nonsenescent adult cells makes it a potential target to eliminate senescent cells. FOXO4 binds p53 and prevents it from inducing the apoptosis of senescent cells. For this reason, impairing this FOXO4-TP53 interaction may be useful to activate apoptosis in these cells. FOXO4-DRI is a cell-permeable peptide that includes part of the p53-interaction domain of FOXO. This peptide could compete with endogenous FOXO4 for p53, impairing the FOXO4-p53 interaction, and inducing apoptosis in senescent cells. Several studies have shown that FOXO4-DRI selectively eliminates senescent cells, without affecting nonsenescent ones. Another peptide recently developed by molecular modelling is ES2, which has the same function as the previously described. The authors demonstrated that combination therapy of ES2 and a Braf inhibitor results in apoptosis and a survival advantage in mouse models of Braf mutant melanoma and reduced senescent cells in ageing mice. Furthermore, they showed that ES2 is effective at eliminating both normal and cancer senescent cells.” (2)

"Induction of senescence by the anticancer treatment of tumor cells and the effects of senescent cells and secreted SASP in cancer. Senotherapies in cancer try to remove chemotherapy-induced senescent cells and/or block deleterious SASP." (2)

Exploiting senescence for the treatment of cancer

“The forkhead box protein O4 (FOXO4)-DRI peptide interferes with the binding of FOXO4 to p53, which occurs in senescent cells, and this activates senolysis." (3)

"To achieve better antitumour responses and to inhibit tumour progression mediated by senescence-associated secretory phenotype (SASP) factors, senescence-inducing therapies can be combined with senolytic treatments. Senolysis can also be mediated by recruitment of immune cells by the SASP factors produced by senescent cells. Detection of senescent cells within the patient’s tumour is important to assess the efficacy of pro-senescence therapies. One way this can potentially be achieved is by a PET-CT scan using an ¹⁸F-labelled β-galactosidase tracer (¹⁸F-β-gal). As potential non-invasive approaches, senescence-associated proteins or metabolites can be detected to measure senescence burden in blood. These technologies provide possibilities to evaluate the efficacy of pro-senescence–senolytic treatment responses and could help guide future clinical trials. NK, natural killer." (3)

Effects of senescent cells in tumours

"The SASP factors suppress cancer in part by reinforcing the senescent growth arrest and/or by promoting immune surveillance. Oncogene-induced and therapy-induced senescent cells secrete the inflammatory cytokine IL-1α, which is a crucial SASP initiator and regulator. IL-1α triggers an autocrine inflammatory response through activation of NF-κB, which leads to the transcription of IL-6 and IL-8. Subsequently, these inflammatory cytokines reinforce senescence proliferation arrest through increased production of reactive oxygen species and a sustained DNA damage response, particularly in oncogene-induced senescent cells. Furthermore, IL-1α also mediates paracrine senescence in neighbouring cells to suppress tumour progression. Moreover, IL-1α, IL-6 and IL-8 mediate the recruitment of M1-like macrophages, T helper 1 cells and natural killer (NK) cells to the tumour microenvironment. These infiltrating immune cells drive the elimination of senescent cancer cells and might also eliminate non-senescent cancer cells through a bystander effect, although this is not yet proven. In addition, immune cells, such as T helper 1 cells, can also trigger senescence in cancer cells through the secretion of inflammatory cytokines."

"Although the SASP of senescent cancer cells is initially tumour suppressive by reinforcing growth arrest and promoting immune clearance, it is suggested to be mostly detrimental in the long term. Early evidence for this notion was found two decades ago by an in vivo study demonstrating increased proliferation and tumorigenesis of both premalignant and malignant epithelial cells when co-injected with human senescent fibroblasts in mice; the SASP was an important contributor to this effect. Another study observed that MMPs secreted by senescent human fibroblasts were of primary importance in promoting tumorigenesis. These prominent SASP factors are involved in the processing and degradation of the extracellular matrix, which can promote cancer cell growth and invasion. In addition, MMPs also promote the release of many other cytokines and growth factors supporting tumorigenesis such as vascular endothelial growth factor (VEGF), which promotes tumour-driven angiogenesis. Furthermore, senescent cells also secrete the chemokine CXCL1, which promotes tumour growth.""Perhaps even more surprisingly, several studies have demonstrated that senescence-inducing therapies are associated with complex reprogramming that could eventually drive stemness in both tumour and normal cells. Moreover, remaining senescent cancer cells that are not cleared by the immune system can spontaneously escape proliferation arrest under certain circumstances and re-enter the cell cycle. Another study observed that oncogene-induced senescent cells could also re-enter the cell cycle, particularly by restoring telomerase activity through de-repression of the telomerase reverse transcriptase (TERT) gene. Importantly, senescent cells that resume growth have a WNT-dependent enhanced growth and tumour initiating potential. This senescence-associated stemness results in a highly aggressive tumour, driven by WNT pathway activation independent of the WNT ligand via the SASP and is found to be enriched in relapsed tumours. Moreover, expression of β-catenin in pituitary stem cells provokes a signature of senescence and SASP and can induce craniopharyngioma tumours in a paracrine fashion. Importantly, mice with reduced senescence burden and SASP responses showed decreased tumorigenic potential, indicating that the SASP may drive tumour induction."

"Taken together, the role of cellular senescence in tumours and the outcome of senescence-inducing therapies are complex and often unpredictable, mainly because of the dual role of the SASP. The effect of the SASP is highly dependent on context and cell type and variable during the different stages of cancer progression." (3)

Immune response-mediated senolysis.

"To study the possibility that increased immune surveillance through therapy targeting the immune checkpoint PD1 can be senolytic, trametinib and palbociclib were combined to induce senescence in a KRAS-mutant pancreatic ductal adenocarcinoma mouse model. Besides direct senolytic effects of anti-PD1 antibody therapy in combination with palbociclib and trametinib, the study also demonstrated an impact of therapy-induced senescence on improving tumour vasculature function through SASP-facilitated vascular remodelling. This response increases blood vessel density and permeability, leading to enhanced uptake and activity of the chemotherapeutic agent gemcitabine, which is the standard-of-care therapy for pancreatic cancer patients. Moreover, in the tumour microenvironment, SASP induction facilitates increased endothelial cell expression of VCAM1, a cell surface protein that stimulates lymphocyte adhesion and extravasation into tissues Other SASP components, such as the pro-angiogenic factor VEGF and the pro-inflammatory cytokines and chemokines CCL5, CXCL1 and IL-6, also facilitate an increase of CD8 T cell infiltration into tumours and improve the efficacy of checkpoint blockade anti-PD1 therapy. These data are intriguing and establish a link between therapy-induced SASP, vascular remodelling and improvement of T cell-based immunotherapies. The previous work of the same group using a mouse Kras mutant lung cancer model identified an NK cell surveillance programme without involvement of T cells. Both studies used mouse models driven by the same KRAS and Trp53 mutations and senescence inducers. However, immune surveillance programmes can be somewhat different between tissues. The lungs are generally rich in NK cells but these cells are scarcer in the pancreas, which may explain the different involvement of immune cells. Differences in the SASP produced by different cancer types may further contribute to tissue-specific differences in immune surveillance. Moreover, the effects of pro-senescence therapy on immune responses and anti-PD1 efficacy has been demonstrated in multiple preclinical studies in various cancer types." (3)

Chemotherapies and radiotherapies

"Despite the ability of malignant tumours to evade senescence, they can still be forced to enter a senescent state using therapeutics leading to therapy-induced senescence. Conventional anticancer therapeutics, such as chemotherapy or radiotherapy, are known to induce senescence in cancer cells.

Low doses of chemotherapy particularly trigger a senescent cell state in human cancer cells, while apoptosis is induced at higher doses. This finding might explain why often only a subset of cancer cells become senescent in response to conventional chemotherapies or radiotherapies as the senescence response is only triggered in a specific window of DNA damage. Mechanistically, many chemotherapies cause DNA damage in cancer cells, which triggers senescence through ATM–CHK2 and ATR–CHK1 kinase-mediated activation of the interconnected p53–RB pathways. Topoisomerase I and II inhibitors, such as doxorubicin, etoposide and camptothecin, are widely used for the treatment of a variety of cancer types and have been shown to dysregulate re-ligation of DNA strands after supercoil unwinding. This leads to massive DNA damage and increased expression of p53 and its downstream targets CDKN1A and PAI1 (also known as SERPINE1), subsequently inducing senescence. Platinum-based compounds, such as cisplatin, carboplatin and oxaliplatin, also induce extensive DNA damage through DNA cross-linking, resulting in senescence induction. Similarly, alkylating agents, such as temozolomide, dacarbazine and busulfan, form DNA crosslinks by reacting with atoms in DNA, triggering a DNA damage-mediated senescence response. Microtubule inhibitors, such as paclitaxel, docetaxel and vinca alkaloids, dysregulate the normal microtubule spindle dynamics to impair metaphase–anaphase transition and arrest the cells at mitosis. This cell cycle dysfunction may also cause extensive DNA damage and trigger a p53–p21-facilitated senescence response. Methotrexate and gemcitabine both induce genotoxic stress by blocking DNA synthesis, thereby inducing cellular senescence. It is important to keep in mind that, while quite a few existing chemotherapeutics have some ability to induce senescence, the apoptotic response is dominant in most cancers. As such, most chemotherapies are unable to induce senescence in a significant fraction of cancer cells in vivo.

Radiotherapy is applied broadly for the treatment of multiple cancer types. This anticancer treatment can induce an irreparable DNA damage response that activates ATM or ATR and p53–p21 pathway-mediated apoptosis and cellular senescence. As, unlike chemotherapy, the treatment can be applied locally, there is less collateral damage to normal tissues and, consequently, potentially also less secondary cancer. Nevertheless, the tissue surrounding the cancer can show an increase in senescent cell burden, resulting in an array of local side effects, including immunosuppressive effects.

It will be crucial to better understand how the SASP produced by senescent cancer cells impacts the interaction between senescent cancer cells and the immune system. Some early data point towards a synergy between pro-senescence therapy and checkpoint immunotherapy. It is important to keep in mind that there is a publication bias for experiments that yield positive results. It is therefore possible that not all pro-senescence therapies and not all cancer types will benefit from combination with checkpoint immunotherapy." (3)

Cellular senescence in malignant cells promotes tumor progression in mouse and patient Glioblastoma

"Senescent cell’s partial removal increases the survival of GBM-bearing mice." (4)

"To confirm the tumor-promoting function of senescent cells, we further studied GBM mice carrying the p16-3MR transgene. On average, about 2% of the tumor area was comprised of SA-β-gal cells in WT+GCV GBMs, which corresponds to senescent category one as we defined using patient gliomas.

We next performed bulk RNA sequencing (RNAseq) of the tumors with or without senescent cells. In agreement with the inter-tumoral heterogeneity of patient GBMs, heat maps of the bulk RNAseq data revealed inter-tumoral heterogeneity of mouse GBMs independent of the treatment. Gene set enrichment analysis of p16-3MR+GCV GBMs compared with WT+GCV GBMs revealed an upregulation of cell cycle components, a downregulation of pathways involved in cancer (Notch signaling, mTORC1 signaling, epithelial–mesenchymal transition, angiogenesis), and modulation of the immune system (TNFA signaling via NFKB, Interferon responses, Il2-Stat5 signaling).

Finally, GSEA revealed a significant downregulation of senescence pathways. SASP genes whose expression was significantly decreased in p16-3MR+GCV compared with WT+GCV GBMs included Fn1, Plau, Timp1, Ereg, and Bmp2, the qPCR analysis further validated Ereg decrease. These SASP genes encode growth factors and extracellular matrix components or remodelers.

Collectively our data show that at the late timepoint, when mice were sacrificed due to tumor burden, there was an increased survival of GBM-bearing mice associated with the partial removal of p16 ^Ink4a senescent cells, therefore pointing to the tumor-promoting action of senescent cells during gliomagenesis." (4)

References

(1) Putavet, Diana, et al. “Abstract P1-19-02: Repurposing the FOXO4 Senolytic against Triple-Negative Breast Cancer.” Cancer Research, vol. 82, no. 4_Supplement, 15 Feb. 2022, pp. P1-1902-P119-02, aacrjournals.org/cancerres/article/82/4_Supplement/P1-19-02/680494, 10.1158/1538-7445.sabcs21-p1-19-02.

(2) Sánchez-Díaz, Laura, et al. “Senotherapeutics in Cancer and HIV.” Cells, vol. 11, no. 7, 1 Jan. 2022, p. 1222, www.mdpi.com/2073-4409/11/7/1222/htm#B1-cells-11-01222, 10.3390/cells11071222.

(3) Wang, Liqin, et al. “Exploiting Senescence for the Treatment of Cancer.” Nature Reviews Cancer, 3 Mar. 2022, 10.1038/s41568-022-00450-9.

(4) Salam, Rana, et al. “Cellular Senescence in Malignant Cells Promotes Tumor Progression in Mouse and Patient Glioblastoma.” Nature Communications, vol. 14, no. 1, 27 Jan. 2023, p. 441, www.nature.com/articles/s41467-023-36124-9, 10.1038/s41467-023-36124-9.

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