Genome-wide CRISPR/Cas9 screen identified MCL1 as a senolytic target for clearing palbociclib-induced senescent and PD-L1-positive cells in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. Although the use of small molecule drugs or targeted drugs has shown significant efficacy in the treatment of CRC, the drug resistance after treatment and the high recurrence and metastasis rate are the key obstacles affecting the success rate of treatment and survival of patients. Cellular senescence constitutes an important barrier to tumor progression. Senescent tumor cells and stromal cells are among the reasons for cancer treatment resistance. Different senescent programs can exert inhibitory or promotional effects on CRC. In serrated adenomas of colon, the senescence induced by intrinsic oncogenes serves as a threshold that precancerous lesions must traverse to develop into cancer. And the exposing of anti-cancer treatment, such as chemotherapy and radiotherapy, some cells also enter a senescent state, presenting a stable cell cycle arrest and senescence-associated secretory phenotype (SASP). SASP can activate immune surveillance but also contribute to the maintenance of cellular senescence microenvironment to help the CRC progression. Hence, in the pursuit of effective CRC treatment strategies, the issue of senescent cells is inevitable. By targeting features of senescent cells, such as upregulated anti-apoptotic signaling, altered metabolic signaling, and differential SASP secretion, depletion of senescent cells could be a promising strategy for the treatment of CRC. This review summarizes the endogenous and exogenous factors leading to cell senescence in CRC, as well as drug mechanisms, and focuses on the research progress of senescent tumors and stromal cells in CRC. Eventually, we discuss the strategies for CRC senescent cells after anti-cancer treatment to provide some theoretical basis and direction for retarding the malignant progression and recurrence of CRC.

IL-4/IL-13 signaling suppresses ABT-263-induced apoptosis in senescent human fibroblasts.

Background and AimsThe initiation of cellular senescence in response to protumorigenic stimuli counteracts malignant progression in (pre)malignant cells. Besides arresting proliferation, cells entering this terminal differentiation state adopt a characteristic senescence-associated secretory phenotype (SASP) which initiates alterations to their microenvironment and effects immunosurveillance of tumorous lesions. However, some effects mediated by senescent cells contribute to disease progression. Currently, the exploration of senescent cells' impact on the tumor microenvironment and the evaluation of senescence as possible target in colorectal cancer (CRC) therapy demand reliable detection of cellular senescence in vivo. Therefore, specific immunohistochemical biomarkers are required. Our aim is to analyze the clinical implications of senescence detection in colorectal carcinoma and to investigate the interactions of senescent tumor cells and their immune microenvironment in vitro and in vivo.MethodsSenescence was induced in CRC cell lines by low-dose-etoposide treatment and confirmed by Senescence-associated β-galactosidase (SA-β-GAL) staining and fluorescence activated cell sorting (FACS) analysis. Co-cultures of senescent cells and immune cells were established. Multiple cell viability assays, electron microscopy and live cell imaging were conducted. Immunohistochemical (IHC) markers of senescence and immune cell subtypes were studied in a cohort of CRC patients by analyzing a tissue micro array (TMA) and performing digital image analysis. Results were compared to disease-specific survival (DSS) and progression-free survival (PFS).ResultsVarying expression of senescence markers in tumor cells was associated with in- or decreased survival of CRC patients. Proximity analysis of p21-positive senescent tumor cells and cytotoxic T cells revealed a significantly better prognosis for patients in which these cell types have the possibility to directly interact. In vitro, NK-92 cells (mimicking natural killer T cells) or TALL-104 cells (mimicking both cytotoxic T cells and natural killer T cells) led to dose-dependent specific cytotoxicity in >75 % of the senescent CRC cells but <20 % of the proliferating control CRC cells. This immune cell-mediated senolysis seems to be facilitated via direct cell-cell contact inducing apoptosis and granule exocytosis.ConclusionCounteracting tumorigenesis, cellular senescence is of significant relevance in CRC. We show the dual role of senescence bearing both beneficial and malignancy-promoting potential in vivo. Absence as well as exceeding expression of senescence markers are associated with bad prognosis in CRC. The antitumorigenic potential of senescence induction is determined by tumor micromilieu and immune cell-mediated elimination of senescent cells.

ABSTRACTChemotherapy‐induced senescence‐associated tumor microenvironment (S‐TME) facilitates colorectal cancer (CRC) progression. This study elucidates the mechanism by which Tianma granule (TMG), a traditional Chinese medicine formula, remodels the S‐TME and inhibits CRC, specifically investigating the role of the miR‐29a‐5p/P53 axis. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) identified 18 bioactive components in TMG, and network pharmacology highlighted P53 as a core target. Functional assays, including Cell Counting Kit‐8 (CCK‐8), β‐galactosidase staining, flow cytometry, wound‐healing, and Transwell migration tests, were conducted using doxorubicin (DOX)–induced senescent human umbilical vein endothelial cells (HUVECs) and CRC lines. TMG suppressed CRC cell proliferation, motility, and invasiveness while promoting apoptosis. TMG reduced P53/cyclin‐dependent kinase inhibitor 1A (P21) and senescence‐associated secretory phenotype (SASP) factors (IL‐6, IL‐8, CCL20), while upregulating miR‐29a‐5p in senescent HUVECs. miR‐29a‐5p inhibition enhanced senescence and increased P53/P21/SASP, whereas P53 silencing lowered P21 and decreased miR‐29a‐5p, indicating mutual regulation. In azoxymethane/dextran sulfate sodium (AOM/DSS)‐CRC mice, TMG reduced tumor burden and improved survival, accompanied by lower P53/P21 and restored miR‐29a‐5p in tissues. TMG remodels the chemotherapy‐induced S‐TME and suppresses CRC progression by modulating the miR‐29a‐5p/P53 axis, enhancing apoptosis in senescent cells, and counteracting S‐TME‐mediated tumor growth and metastasis. This highlights TMG's therapeutic potential.

Simple SummaryChemotherapy can lead to cellular senescence in tumor cells. Here we demonstrate that oxaliplatin induces senescence in p53-proficient colorectal cancer (CRC) cells and leads to the G2-phase arrest in all lines studied (HCT116p53+/+, HCT116p53−/−, LoVo, SW48, and SW480). At early times the p53-competent lines activate p53 and p21CIP1, however, at later times, only LoVo cells showed sustained p53/p21CIP1 activation, accompanied by a strong induction of senescence and senescence-associated secretory phenotype (SASP) factors. Opposite to LoVo, the p53/p21CIP1 response and senescence induction is much weaker in the other p53-proficient cells, due to p14ARF deficiency. LoVo cells express p14ARF protein and siRNA-mediated knockdown of p14ARF significantly reduces sustained p53/p21CIP1 activation and senescence. Vice versa, ectopic expression of p14ARF enhances oxaliplatin-induced senescence in SW48 and SW480 cells. Our data show that oxaliplatin-induced senescence in CRC cells depends on p53 proficiency; however, a significant induction can only be observed upon p14ARF-mediated p53 stabilization.Senescence is an important consequence of cytostatic drug-based tumor therapy. Here we analyzed to which degree the anticancer drug oxaliplatin induces cell death, cell cycle arrest, and senescence in colorectal cancer (CRC) cells and elucidated the role of p53. Oxaliplatin treatment resulted in the G2-phase arrest in all CRC lines tested (HCT116p53+/+, HCT116p53−/−, LoVo, SW48 and SW480). Immunoblot analysis showed that within the p53-competent lines p53 and p21CIP1 are activated at early times upon oxaliplatin treatment. However, at later times, only LoVo cells showed sustained activation of the p53/p21CIP1 pathway, accompanied by a strong induction of senescence as measured by senescence-associated β-Gal staining and induction of senescence-associated secretory phenotype (SASP) factors. Opposite to LoVo, the p53/p21CIP1 response and senescence induction was much weaker in the p53-proficient SW48 and SW480 cells, which was due to deficiency for p14ARF. Thus, among lines studied only LoVo express p14ARF protein and siRNA-mediated knockdown of p14ARF significantly reduced sustained p53/p21CIP1 activation and senescence. Vice versa, ectopic p14ARF expression enhanced oxaliplatin-induced senescence in SW48 and SW480 cells. Our data show that oxaliplatin-induced senescence in CRC cells is dependent on p53 proficiency; however, a significant induction can only be observed upon p14ARF-mediated p53 stabilization.

ObjectivesEndothelial cell dysfunction during aging is a key driver of vascular aging and related diseases; however, effective strategies to selectively eliminate senescent endothelial cells and restore vascular function remain lacking. FOXO4-DRI, a novel peptide-based intervention, specifically disrupts the interaction between FOXO4 and P53, thereby inducing apoptosis in senescent cells. This study innovatively focuses on the mechanism by which FOXO4-DRI induces apoptosis in senescent endothelial cells, demonstrating that it functions by activating the p53/BCL-2/Caspase-3 signaling pathway to promote selective apoptosis of these cells. FOXO4-DRI significantly improves vascular function and delays vascular aging. These findings not only enrich the molecular understanding of senescent cell clearance but also provide a novel strategy for precise targeting of endothelial cell senescence in therapeutic applications.Materials and methodsThis study aims to analyze the vascular function and aging status of the aorta in naturally aged mice and progeroid model mice following FOXO4-DRI injection. Additionally, it investigates changes in endothelial cell function in senescent endothelial cells induced by oxygen-glucose deprivation (OGD), as well as the protein expression and interaction in the FOXO4-P53 signaling pathway. To assess the impact of FOXO4-DRI on endothelial cell senescence, the senescent endothelial cells were treated with FOXO4-DRI, followed by immunofluorescence and Western blotting experiments.ResultsInjection of FOXO4-DRI in both naturally aged and induced aging mice effectively suppressed aortic aging and improved aortic function. Additionally, we found that FOXO4-DRI alleviates endothelial cell senescence induced by OGD, thereby enhancing endothelial cell function. Through co-immunoprecipitation (CO-IP) experiments, we discovered that FOXO4-DRI prevents the binding of FOXO4 to P53, facilitating the phosphorylated P53 nuclear exclusion, which subsequently trigger BAX and cleaved caspase-3, leading to the apoptosis of senescent cells. Ultimately, this mechanism achieves the goal of inhibiting vascular aging.ConclusionFOXO4-DRI promotes the nuclear export of phosphorylated P53 by inhibiting the binding of FOXO4 to P53 in endothelial cells, thereby facilitating the apoptosis of senescent endothelial cells and alleviating aging.

Senescence, largely initiated by unrepaired DNA damage, is initially sustained by upregulated p53. Although senescent cells enter proliferative arrest, early p53 supression may result in senescent cells re-entering the cell cycle. Senescent cells exhibit a senescence-associated secretory phenotype (SASP) that impacts the cell microenvironment, often promoting cell transformation. SASP molecular composition may be cell-specific and dependant on the type of SASP-producing cell. As growth hormone (GH) is induced by DNA damage, we elucidated whether GH is induced in senescent cells. Non-pituitary GH (npGH) synthesized locally in peripheral tissues is identical to endocrine GH1 produced by the pituitary and acts through autocrine/paracrine mechanisms via the widely expressed GH receptor that recognizes both pituitary GH and npGH ligands. We show that npGH is induced in aging human colon tissue, and in human non-tumorous colon cells and in human 3-dimensional intestinal organoids in response to oncogene-, therapy-, and/or replicative-induced senescence. Furthermore,DNA-damage-induced npGH is secreted from senescent cells, constituting a SASP component. In senescent cells, DNA damage is not repaired with fidelity, and we show that induced npGH suppresses DNA damage responses by attenuating phosphorylation of ATM, DNA-PKc, p53 and Chk2, resulting in p53 suppression and accumulation of damged DNA. Autocrine npGH also triggers senescent cell proliferation with increased Ki67 and BrdU incorporation. As proliferating cells with accumulated unrepaired DNA damage may acquire oncogenic mutations,we assessed npGH actions on cell transformation. We show that senescent colon cells expressing npGH form colonies in soft agar,while GH depletion by shRNA downregulates Ki67 and decreases colony formation and size, suggesting that npGH enables senescent cell transformation. Consistent with a SASP function, induced paracrine npGH also suppresses the p53/p21 pathway, triggering proliferation and exacerbates DNA damage in neighboring non-senescent cells. To further explore mechanisms underlying npGH induction we tested the role of the SASP chemokine CXCL1 which attracts immune effectors to eliminate senescent cells. CXCl1 is shown to induce npGH in senescent hNCC and in intestinal organoids, while GH, in turn, suppresses CXCL1, likely by inhibiting NFκB, a CXCL1 transcription factor. Both colon CXCL1 and NFκB are more abundant in GH-receptor knockout mice devoid of GH signalling, while mice bearing GH-secreting xenografts exhibit decreased colon CXCL1 abundance. Conclusions: The results elucidate a heretofore unappreciated GH action, whereby npGH, as a SASP component, attenuates senescent cell elimination by inhibiting CXCL1, and contributes to a tissue microenvironment favoring age-associated DNA damage accumulation and epithelial cell transformation. Citation Format: Vera Chesnokova, Svetlana M. Zonis, Robert Barrett, Shlomo Melmed. Growth hormone as a SASP component [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5683.

In colorectal cancer, the transition of cancer cells from an adenoid to a collective invasion morphology, ultimately leading to a partial epithelial-mesenchymal transition phenotype at the invasive front, has been observed. Through comprehensive gene expression analysis, we identified distinct gene expression patterns between cancer cells at the invasive front and those in the central region, with cellular senescence playing a pivotal role in this transition. Trajectory analysis revealed a gradual transformation of non-senescent tumor cells in the central region into senescent cells at the invasive front, accompanied by altered gene expression patterns. Furthermore, we observed a progressive increase in the expression of CXCL12 and CSF1 at the invasive front. Senescent tumor cells were found to impair the directional migration of CD8+ T cells by secreting high concentrations of CXCL12. Additionally, CSF1 secreted by senescent tumor cells promoted the differentiation of monocytes into M2 macrophages, which subsequently inhibited CD8+ T cell activation. Importantly, inhibiting CXCL12 secretion from senescent tumor cells enhanced T cell infiltration and resulted in reduced tumor number and size. These findings highlight the generation of a cytokine barrier by senescent tumor cells, which serves to protect non-senescent tumor cells from immune attack. Moreover, this study identifies senescent tumor cells as a potential target for overcoming immunotherapy resistance in colorectal cancer. The development of interventions that disrupt the cytokine barrier and enhance T cell infiltration holds promise for improving the efficacy of immunotherapeutic approaches in the treatment of colorectal cancer. Citation Format: Tae Jun Park, Soon Sang Park. The senescent tumor cells evade the immune system by secreting SASPs in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B026.

Cellular senescence is a double-edged sword in cancer biology, initially acting as a tumor-suppressive mechanism but later contributing to cancer progression and therapy resistance. Senescent cells, characterized by stable cell cycle arrest, secrete a complex array of bioactive molecules known as the senescence-associated secretory phenotype (SASP), which fosters chronic inflammation, disrupts tissue architecture, and promotes tumorigenesis through paracrine signaling. Accumulation of these cells in the tumor microenvironment can enhance malignancy, drive metastasis, and impair treatment outcomes. Senotherapeutics, have emerged as promising strategies for targeting senescent cells in cancer therapy. These agents selectively induce apoptosis in senescent cells while preserving normal tissues, representing a paradigm shift in oncology. Senotherapeutics can function as standalone treatments by clearing senescent tumor cells or as adjuvants to chemotherapy and radiotherapy, effectively eliminating residual therapy-induced senescent cells that may contribute to relapse. This dual approach allows for reduced treatment toxicity, improved therapeutic efficacy, and decreased tumor recurrence. Furthermore, targeting non-cancerous senescent cells may help suppress inflammation-driven tumorigenesis, slow disease progression, and enhance patient outcomes. Despite their promise, challenges remain in optimizing senotherapeutic strategies, identifying precise biomarkers, and minimizing off-target effects. This review explores the mechanisms of cellular senescence, its role in tumor dynamics, and the potential of senotherapeutics as a novel adjunct in cancer treatment. By integrating senotherapeutics with existing modalities, the field moves closer to more effective, personalized cancer interventions, warranting further preclinical and clinical investigation.

Ganoderma lucidum (G. lucidum) is a famous medicinal mushroom that has been reported to prevent and treat a variety of diseases. Different extractions from G. lucidum have been used to manage age-related diseases, including cancer. Nevertheless, the senolytic activity of G. lucidum against senescent cancer cells has not been investigated. Although cellular senescence causes tumor growth inhibition, senescent cells promote the growth of the neighboring tumor cells through paracrine effects. Therefore, the elimination of senescent cells is a new strategy for cancer treatment. In this study, senescence was triggered in HCC cells by the chemotherapeutic agent Adriamycin (ADR), and subsequently, cells were treated with TC to assess its senolytic activity. We found for the first time that the triterpenoid complex (TC) from G. lucidum had senolytic effect, which could selectively eliminate adriamycin (ADR)-induced senescent cells (SCs) of hepatocellular carcinoma (HCC) cells via caspase-dependent and mitochondrial pathways-mediated apoptosis and reduce the levels of senescence markers, thereby inhibiting the progression of cancers caused by SCs. TC could block autophagy at the late stage in SCs, resulting in a significant activation of TC-induced apoptosis. Furthermore, TC inhibited the senescence-associated secretory phenotype (SASP) in SCs through the inhibition of NF-κB, TFEB, P38, ERK, and mTOR signaling pathways and reducing the number of SCs. Sequential administration of ADR and TC in vivo significantly reduced tumor growth and reversed the toxicity of ADR. A triterpenoid complex isolated from G. lucidum may serve as a novel senolytic agent against SCs, and its combination with chemotherapeutic agents may enhance their antitumor efficacy.

Senescent cells accumulate in aged tissue and are causally linked to age-associated tissue degeneration. These non-dividing, metabolically active cells are highly secretory and alter tissue homeostasis, creating an environment conducive to metastatic disease progression. IL-1α is a key senescence-associated (SA) proinflammatory cytokine that acts as a critical upstream regulator of the SA secretory phenotype (SASP). We established that SA shifts in steady-state H2O2 and intracellular Ca(2+) levels caused an increase in IL-1α expression and processing. The increase in intracellular Ca(2+) promoted calpain activation and increased the proteolytic cleavage of IL-1α. Antioxidants and low oxygen tension prevented SA IL-1α expression and restricted expression of SASP components IL-6 and IL-8. Ca(2+) chelation or calpain inhibition prevented SA processing of IL-1α and its ability to induce downstream cytokine expression. Conditioned medium from senescent cells treated with antioxidants or Ca(2+) chelators or cultured in low oxygen markedly reduced the invasive capacity of proximal metastatic cancer cells. In this paracrine fashion, senescent cells promoted invasion by inducing an epithelial-mesenchymal transition, actin reorganization, and cellular polarization of neighboring cancer cells. Collectively, these findings demonstrate how SA alterations in the redox state and Ca(2+) homeostasis modulate the inflammatory phenotype through the regulation of the SASP initiator IL-1α, creating a microenvironment permissive to tumor invasion.

Senescence has been shown to prevent and promote tumorigenesis. [1, 2] These results are not so paradoxical. To develop and maintain, organisms rely on cellular growth and cell division. Each of these processes is spatiotemporally finely tuned and both are tightly coordinated to ensure organism homeostasis throughout life. As organism aged, deregulations of these processes appear leading to hyperplastic or degenerating diseases, such as cancer and Alzheimer disease, respectively [3]. Interestingly, these two aged-related diseases have been linked to a cellular response that yet, uncouples cellular growth from cell division: senescence. Senescence is a natural cellular response that can be triggered by various stimuli, such as telomere shortening, oncogenic stresses or unrepaired DNA damages [4]. Senescent cells grow but do not divide so that they are enlarged and restricted in number. In addition, as they do not proliferate due to the irreversible cell cycle arrest, they do not differentiate. Thus senescence modifies tissue homeostasis by profoundly impacting tissue architecture both physically and biologically. Such disorganisation leads to alteration of cell contacts thereby re-wiring cellular communication. To communicate, cells use physical interactions and diffusible factors. In that context, it is interesting to observe that senescent cells often release factors such as cytokines or growth factors. This is known as senescence associated secretory phenotype (SASP) [5]. Recently Acosta et al have shown that the TGFβ pathway mediates paracrine senescence in SASP and that this pathway and the BMP pathway are upregulated in such senescent cells [6]. Interestingly, these two pathways are involved in tissue morphogenesis during organism development. It is therefore tempting to suggest that one of the outcomes of senescence is tissue re-organisation, achieved via cell communication, to reach new homeostasis upon cellular stress. As a matter of fact, studies of senescent cancer cells suggest so. First, senescence has been shown to act as an anti-cancer barrier, both physically and biologically in preneoplastic tissue [1]. Secondly, it has been shown to promote tumorigenesis by favouring the emergence of cancer stem-like cells (CSLCs) [7]. CSLCs are rare quiescent cells. They niche in heterogeneous tumors and have, in contrast to the bulk tumor cells but similarly to normal stem cells, the ability to self renew and to differentiate. Thus, if tissue has to be re-organised upon senescence to gain minimal homeostasis for functioning, new cells have to emerge and differentiate. This can be achieved by stimulation of CSLCs by SASP factors released from senescent cancer cells. Of note, it remains unclear why CSLCs, unlike normal stem cells, do not senesce. In relation to their role in tissue architecture, it has been described that CSLCs preferentially develop, within the tissue mass, under hypoxic conditions. Interestingly, hypoxia has been shown to inhibit mTOR, which converts quiescent cells into senescent cells [8]. If experimentally verified, hypoxia could reinforce the intrinsic resistance of CSLCs by maintaining their quiescent state, while inhibiting mTOR and geroconversion of CSLCs from quiescence to senescence. It therefore appears, at least in pathological cancer tissue, that senescence, and SASP in particular, could play a pivotal role in tissue re-organisation upon cellular stress. As a consequence, depending on the cancer stage, i.e. to which extend tissue has to be re-organised upon cancer invasion, senescence could be pro or anti tumorigenic. As to whether this role in tissue re-organisation also occurs in non-pathological tissue remains to be investigated. Tissue re-organisation by senescence implies cellular communication through SASP factors. Therefore it will be interesting to investigate if senescence is accompanied by secretion of SASP factors in unicellular organisms. If not, this will strongly argue for a role of SASP in maintaining tissue homeostasis, via tissue re-organisation, in multicellular organisms.

Communication between cells is a vital process that governs cellular organization and coordination in an organism. Thus, multiple mechanisms of cell communication have evolved to respond to all the needs of an organism. The modes of cell-cell communication range from the ones that require direct contact between cells, such as cytoplasmic bridges (CBs) and gap junctions, to others that may take place over long distances throughout the organism, such as hormone signaling. Precise integration of data acquired from all these different modes of communication maintains tissue homeostasis and allows optimal adaptation of an organism to various stresses. Cellular senescence represents one of the outcomes of cellular response to stress.1 Senescent cells execute essential functions in different physiological and pathophysiological conditions. They are present in pre-malignant lesions, sites of tissue damage, aging tissues and even during embryonic development. In all these places senescent cells communicate with cells in their surroundings and modulate the function of these cells.2-5 The effect of senescent cells on nearby cells is commonly attributed to the secretion of cytokines, chemokines and matrix metalloproteinases.4,6 We have recently demonstrated that in addition to secretion, senescent cells affect neighboring cells by direct intercellular protein transfer (IPT).2 Proteins from senescent cells are directly transferred to recipient neighboring cells, such as immune and cancer cells, triggering activation of signaling pathways in these cells, ultimately leading to changes in cellular behavior. We have detected IPT from senescent cells and demonstrated that these cells form CBs with other cells, including immune cells, cancer and non-cancer epithelial cells.2 CBs, which in some conditions are called tunneling nanotubes, are open on both edges and allow transfer of cytoplasmic content to neighboring cells. Interestingly, we also identified mitochondria and lysosomes in CBs, implying that organelles might be transferred between the cells. Transfer of these organelles was indeed identified in several other systems. Since dysfunctional mitochondria are thought to play a role in cellular senescence, it can be speculated that transfer of the dysfunctional mitochondria to healthy neighboring cells may induce senescence in these cells. Transfer of cytoplasmic content or whole organelles might serve therefore, for communication of cellular stress. The functional consequences of cell communication of senescent cells through IPT are not completely understood and could be cell type dependent. For instance, we have reported that IPT facilitates elimination of senescent cells by NK cells since transfer of proteins to NK cells correlated with NK cell activation and cytotoxicity. Remarkably, inhibition of CDC42 in senescent cells resulted in decreased IPT and impaired elimination of the senescent cells by NK cells.2 In addition to interaction with NK cells senescent cells also communicate with other components of the innate and adaptive immune systems.2,5,6 Thus, it is possible that senescent cells transfer proteins to other types of immune cells and alter their behavior. To fulfill their function immune cells respond to cytokines and chemokines using receptor-ligand interactions to activate cellular responses (Fig. 1). IPT can serve as an additional mechanism regulating activity of immune cells following contact mediated interaction with resident cells and thereby fine-tune the immune response. Figure 1. Senescent cells communicate with NK cells by multiple mechanisms. Senescent cells influence NK cells by (i) senescent associated secretory phenotype (SASP); (ii) specific receptor ligand interactions; (iii) cytoplasmic bridges. These diverse mechanisms ... Senescent cells can impact tumorigenesis, mainly by secretion of pro-inflammatory cytokines.3,5 Our data demonstrate that senescent cells form CBs with cancer cells, thereby implementing IPT in interaction of senescent and cancer cells. Since the interaction of these cells is long lasting, in contrast to the interaction with NK cells, higher amount and number of proteins could potentially be transferred. The functional impact of IPT from senescent cells to cancer cells is unknown, as it could potentially promote or restrain cancer cells. Senescent cells could transfer cell cycle inhibitors (i.e. p16, p15 and p21) to cancer cells, thereby inhibiting their proliferation. Conversely, cancer cells may also receive organelles and proteins to support their rapid proliferation. In addition to pathological conditions, senescent cells modulate physiological processes, including embryonic development.4 During embryonic development human placental syncytiotrophoblast exhibit features and molecular markers of cellular senescence.7 The syncytiotrophoblast is a multinucleate epithelium which supports fetal growth by creating an interface between maternal and fetal circulation. The syncytiotrophoblast cells communicate with subjacent layer of mono-nucleated cytotrophoblasts to promote division and fusion of cytotrophoblasts into the syncytiotrophoblast. These cells also communicate with immune cells to control immune tolerance at the maternal-fetal interface. Therefore, senescent syncytiotrophoblast cells might also form CBs to facilitate these non-cell autonomous effects which are essential for embryonic development. Overall, IPT from senescent cells represents a pivotal mode of cellular communication with possible physiological outcomes on cancer progression, tissue repair, embryonic development, aging and immune modulation.

Deficient GH signaling results in lifespan extension in murine and human models, while patients with uncontrolled acromegaly and transgenic mice overexpressing GH have a shorter lifespan. Colon polyp development increases with age, and also with GH excess in acromegaly. Aging is characterized by senescent cell accumulation with p53/p21 or p16 upregulation as well as cell cycle arrest and expression of a senescence-associated secretory phenotype (SASP). Senescence is reinforced by the SASP, which comprises pro-inflammatory cytokines, chemokines, growth modulators, angiogenic factors, and matrix metalloproteinases. SASP contributes to pro-aging phenotypes, and depletion or removal of senescent cells increases lifespan by partially protecting from age-related pathologies. Senescence, triggered by genotoxic insult with DNA damage, can be reversed by p53 inactivation; senescent cells with low p53 and unrepaired DNA damage can then re-enter the cell cycle (Beausejour et al, EMBO 2003), potentially resulting in chromosomal instability. We showed GH induction in non-pituitary senescent cells leading to suppressed p53/p21 (Chesnokova PNAS 2013). We now show that, in senescent human colon cells (hNCC) and in 3-dimensional human intestinal organoids, non-pituitary GH (npGH) is a component of SASP. In response to DNA damage, npGH is expressed and secreted locally as measured by RT-PCR, WB, and ELISA. High autocrine/paracrine GH further exacerbate DNA damage and reverses senescent in colon cells, enabling them to re-enter the cell cycle, as evidenced by p53 downregulation and increased Ki67 expression. Senescent colon cells expressing high intracellular GH form colonies in soft agar, indicative of cell transformation and proliferation, while GH deletion by shRNA results in Ki67 downregulation and decreased colony formation and size. The SASP protein CXCL1, a chemo-attractant that also functions to eliminate senescent cells, dose-dependently activates GH in hNCC and in intestinal organoids. GH, in turn, suppresses CXCL1 expression. Consistent with these findings, colon CXCL1 was induced in GHRKO mice, and also in hNCC with abrogated GH signaling by shRNA. Taken together with our finding that GH accumulates in aging normal human colon tissue and co-localizes in cells expressing senescence-associated β-galactosidase, these results suggest that GH, as a SASP component, initiates senescent cell proliferation and transformation. By inhibiting CXCL1, GH also functions to attenuate immune-mediated senescent cell elimination that protects aging tissue from deleterious effects of SASP. These mechanisms may underly an initial step in age-associated epithelial polyp development.

Background: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. More comprehensive studies of key molecular alterations were urgent. Cancer-associated fibroblasts (CAFs) exhibit the senescence-associated secretory phenotype (SASP) which contributes to the progression of cancer through the transcriptomic reprogramming at their senescent states. N7-methylguanosine (m7G), one of the most common RNA modifications, is catalyzed by Methyltransferase-like 1 (METTL1) in human RNA. We therefore investigated the role of METTL1-mediated transcriptomic reprogramming in the crosstalk between senescent CAFs (s-CAFs) and cancer cells. Methods: The SA-β-gal staining and immunofluorescence assays were conducted to characterize the senescent cells. The transwell, wound scratch and organoids assays were preformed to test the abilities of viability, migration and invasion. The concentrations of cytokines and chemokines were determined by Luminex liquid suspension chip. The methylated RNA immunoprecipitation and dot blot assays were performed to characterize the RNA m7G levels in the transcriptome. Results: Through the activation of mTOR signaling, the expression of P16 gene was significantly up-regulated in s-CAFs which was treated by H2O2, accompanied by an increased proportion of SA-β-gal+ cells and altered expression level of METTL1 protein. Luminex liquid suspension chip analysis revealed a significant increase in SASP cytokines such as TNF-α and PDGF-BB in the conditioned media of s-CAFs. Co-culturing CRC cell and organoids with s-CAFs resulted in down-regulation of METTL1 expression and significantly enhanced its invasion and migration abilities, while organoids exhibited increased activity and proliferated more rapidly. TNF-α and PDGF-BB down-regulated the METTL1 expression in CRC cell, while inhibitors targeting TNF-α and PDGF-BB rescued this trend. Interestingly, the regulatory effect on METTL1 of CRC cell by s-CAFs which treated by rapamycin was disappeared. These results indicated that METTL1 modulates RNA m7G modifications influenced by SASP cytokines for promoting CRC metastasis flow. Conclusions: In summary, our findings illustrated that METTL1 plays a role in relaying tumor-promoting signals from s-CAFs in response to SASP cytokines. This ultimately contributes to the tumor-promoting signal flow from s-CAFs. This highlights the importance of understanding the molecular mechanisms underlying the communication between different cell types in the tumor microenvironment. In addition, it also provides a novel strategy for clinical treatment in advanced CRC. Keywords: METTL1, Epigenetic regulation, Senescent CAFs, Tumor microenvironment Citation Format: Jingrong Weng, Xiaolin Wang, Xiaoxia Liu, Yanxin Luo, Huichuan Yu. Senescent cancer-associated fibroblast drives colorectal cancer progression in a METTL1-dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1728.