Senescent Fibroblasts Research Articles

Curculigoside Attenuates Endoplasmic Reticulum Stress-Induced Epithelial Cell and Fibroblast Senescence by Regulating the SIRT1-P300 Signaling Pathway.

The senescence of alveolar epithelial cells (AECs) and fibroblasts plays a pivotal role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a condition lacking specific therapeutic interventions. Curculigoside (CCG), a prominent bioactive constituent of Curculigo, exhibits anti-osteoporotic and antioxidant activities. Our investigation aimed to elucidate the anti-senescence and anti-fibrotic effects of CCG in experimental pulmonary fibrosis and delineate its underlying molecular mechanisms. Our findings demonstrate that CCG attenuates bleomycin-induced pulmonary fibrosis and lung senescence in murine models, concomitantly ameliorating lung function impairment. Immunofluorescence staining for senescence marker p21, alongside SPC or α-SMA, suggested that CCG's mitigation of lung senescence correlates closely with the deceleration of senescence in AECs and fibroblasts. In vitro, CCG mitigated H2O2-induced senescence in AECs and the natural senescence of primary mouse fibroblasts. Mechanistically, CCG can upregulate SIRT1 expression, downregulating P300 expression, enhancing Trim72 expression to facilitate P300 ubiquitination and degradation, reducing the acetylation levels of antioxidant enzymes, and upregulating their expression levels. These actions collectively inhibited endoplasmic reticulum stress (ERS) and alleviated senescence. Furthermore, the anti-senescence effects and mechanisms of CCG were validated in a D-galactose (D-gal)-induced progeroid model. This study provides novel insights into the mechanisms underlying the action of CCG in cellular senescence and chronic diseases, offering potential avenues for the development of innovative drugs or therapeutic strategies.

Exosomes from senescent epithelial cells activate pulmonary fibroblasts via the miR-217-5p/Sirt1 axis in paraquat-induced pulmonary fibrosis

BackgroundParaquat (PQ) is a widely used and highly toxic herbicide that poses a significant risk to human health. The main consequence of PQ poisoning is pulmonary fibrosis, which can result in respiratory failure and potentially death. Our research aims to uncover a crucial mechanism in which PQ poisoning induces senescence in epithelial cells, ultimately regulating the activation of pulmonary fibroblasts through the exosomal pathway.MethodsCellular senescence was determined by immunohistochemistry and SA-β-Gal staining. The expression of miRNAs was measured by qPCR. Pulmonary fibroblasts treated with specific siRNA of SIRT1 or LV-SIRT1 were used to analysis senescent exosomes-mediated fibroblasts activation. Luciferase reporter assay and western blot were performed to elucidated the underlying molecular mechanisms. The effects of miR-217-5p antagomir on pulmonary fibrosis were assessed in PQ-poisoned mice models.ResultsImpairing the secretion of exosomes effectively mitigates the harmful effects of senescent epithelial cells on pulmonary fibroblasts, offering protection against PQ-induced pulmonary fibrosis in mice. Additionally, we have identified a remarkable elevation of miR-217-5p expression in the exosomes of PQ-treated epithelial cells, which specifically contributes to fibroblasts activation via targeted inhibition of SIRT1, a protein involved in cellular stress response. Remarkably, suppression of miR-217-5p effectively impaired senescent epithelial cells-induced fibroblasts activation. Further investigation has revealed that miR-217-5p attenuated SIRT1 expression and subsequently resulted in enhanced acetylation of β-catenin and Wnt signaling activation.ConclusionThese findings highlight a potential strategy for the treatment of pulmonary fibrosis induced by PQ poisoning. Disrupting the communication between senescent epithelial cells and pulmonary fibroblasts, particularly by targeting the miR-217-5p/SIRT1/β-catenin axis, may be able to alleviate the effects of PQ poisoning on the lungs.

Abstract 273: Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression

Abstract Melanoma is the deadliest form of skin cancer, with chronological aging as a major negative prognostic factor due to the influence of intrinsic aging on the tumor microenvironment (TME) to promote tumor initiation and progression. Ultraviolet radiation (UVR) exposure, which is a major risk factor for melanoma, also can cause a physical premature aging phenotype, known as extrinsic photoaging, from chronic UVA irradiation of the underlying dermis. Photoaging of the skin often occurs in tandem with chronological aging, and contributes to similar physical alterations in normal skin structure. However, the molecular mechanisms of how photoaging compares to chronological aging in changing the TME and on tumor progression is still lacking. Here we found that extrinsically photoaged fibroblasts have a higher level of secretion of lysosomal proteases, Cathepsins, which have been shown to be secreted via lysosomal exocytosis and play a functional role in extracellular matrix (ECM) remodeling at neutral PH levels. We found that Cathepsins B and D are secreted at higher levels in both extrinsically photoaged young fibroblasts and in intrinsically aged fibroblasts and upregulated at the endogenous protein level. Cathepsin B has been shown to be highly expressed in metastatic but not primary melanoma patient samples, and cleaves ECM proteins such as fibronectin and tenascin. Immunofluorescent staining of in-vitro fibroblast derived matrices (CDMs) confirmed that photoaging disrupts subsequent deposited extracellular matrix proteins similarly to that of intrinsically aged healthy fibroblasts and these matrix changes influences the growth pattern of melanoma lines plated on these CDMs. Mass spectrometry analysis of secreted proteomics confirmed that aged fibroblasts secrete more cathepsins than intrinsically young fibroblasts, and Cathepsins B and D were also detected in extracellular vesicle (EV) cargo proteomics. Single-Particle Interferometric Reflectance Imaging (SP-IRIS) of EVs showed that UVA irradiation induced a significant increase in the total number of secreted of exosomes and in expression of tetraspanin protein CD63, which plays a role in cargo sorting and has been detected in high levels in the plasma of melanoma patients. We hypothesize that extrinsic photoaging of fibroblasts induces the secretion of exosomes packaged with Cathepsins that play an integral role in ECM remodeling and matrix degradation in a beneficial manner for melanoma invasion. Further studies will assess the influence on secreted cathepsins on melanomagenesis and melanoma invasion through both mechanical and biochemical changes in the TME with in-vitro and in-vivo studies. Citation Format: Vania Wang, Yash Chhabra, Laura Hueser, Ashani Weeraratna. Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression [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 273.

Abstract 1499: Senescent stromal cells drive lung metastases of breast cancer

Abstract In 2022 in the United States, there were nearly 300,000 women diagnosed with breast cancer (BC) with the risk increasing significantly after age 50. The overall 5-year survival rate dramatically decreases, from 99% for patients with stage I, to a devastating 25% for those with stage IV (metastatic) BC, making distal metastasis the leading cause of BC mortality. This mortality rate has not changed in decades with lungs being one of the most common sites for distal metastasis of BC. The BC microenvironment is a complex mix, consisting of tumor cells and numerous nontumor cells including distinct subpopulations of cancer-associated fibroblasts (CAFs): vascular (vCAFs), inflammatory (iCAFs), and myofibroblastic CAFs (myCAFs), which can play pro- or anti-tumor roles. Senescent fibroblasts, which accumulate with age typically express p16INK4A and can modulate the cells around them via the secretion of a cadre of pro-tumorigenic factors including cytokines, chemokines, and extracellular matrix (ECM) modulating enzymes, collectively called the senescence-associated secretory phenotype (SASP). To investigate how p16+ senescent cells impact tumorigenesis, we mated the well-characterized MMTV-PyMT mouse, to the INK-ATTAC (INK) mouse, wherein treatment with AP20187 (AP) selectively eliminates p16+ senescent cells. Preliminary data showed that elimination of p16+ aSMA+ senescent fibroblasts (senCAF) resulted in a significant decrease in primary tumors, lung tumor foci and lung metastatic burden. Further, we found that senescent CAFs (senCAFs) were the only stromal cells eliminated in the PyMT-INK mouse treated with AP, suggesting that this CAF subtype is a key contributor to tumor progression. To determine if senCAFs directly impacted lung metastasis, EO771-LG Luc mammary tumor cells were injected in the tail vein of INKATTAC mice that were treated with AP to eliminate p16+ senCAFs. We found that metastatic tumor burdens were significantly reduced in INK+ versus control mice and immunohistochemical analysis revealed that tumor burden and foci was significantly reduced, suggesting that senCAFs in the lung promoted tumor cell seeding and outgrowth. Citation Format: Anupama Melam, Jiayu Ye, Sheila A. Stewart. Senescent stromal cells drive lung metastases of breast cancer [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 1499.

Abstract 1728: Senescent cancer-associated fibroblast drives colorectal cancer progression in a METTL1-dependent manner

Abstract 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.

Abstract 358: Ferroptosis modulates melanoma progression in an age-specific manner through changes in the tumor microenvironment

Abstract Aging is an independent prognostic factor for melanoma, the most aggressive form of skin cancer. Although immune therapy has shown a dramatic improvement in melanoma treatment, resistance to it still occurs in some patients. Higher oxidative damage in tumor cells as well as lipid and iron accumulation are singular characteristics of the aged tumor microenvironment (TME). Here, we investigate the impact of these characteristics of the aged TME in the promotion of ferroptosis, a caspase independent iron mediated type of cell death, and as possible modulators of the response to checkpoint blockade therapy. Lipid peroxidation levels were assessed through immunohistochemical staining for the marker 4-HNE in tumors of young and aged mice injected intradermally with Yumm1.7 cells.Correlation analysis between transcriptomic data (TCGA-SKCM) was performed to identify common denominators among proteins associated with the aged TME, ferroptosis pathway and IFN-y signaling. Our in vitro experiments consisted of viability assays with combinations of IFN-y, young and aged skin fibroblast conditioned media (CM), arachidonic acid (AA) or ferroptosis inducers such as RSL-3. Protein levels of ferroptosis related markers in melanoma cells treated with fibroblast conditioned media, and in Wnt5a high/low cell lines were analyzed through western blot (WB). Tumors from aged mice showed stronger staining with 4-HNE antibody when compared to young. Wnt5a (present in the aged TME) showed a strong correlation with several ferroptosis related transcripts. WB analysis of Wnt5a high/low lines indicate differences in GPX4, FTL1, SLC3A2, GCH1 and DHODH. Wnt5a levels show an inverse correlation with ferroptosis resistance and anti-ferroptotic proteins. Our data indicate that aged TME favors ferroptosis death in melanoma cells. To understand how ferroptosis impacts tumor growth in young and aged mice, we used a syngeneic tumor model injecting Yumm1.7 cells intra-dermally in mice and treated with a lipid peroxidation inhibitor, Liproxstatin, or an ferroptosis inducer, IKE. Liproxstatin lead to reduced tumor growth only in young mice followed by changes in immune cell populations such as monocytes and macrophages. IFNy induction of these events might be associated with changes in proteins of the pathway or alterations in fatty acid uptake as suggested by lipid accumulation and AA potentiation of its cytotoxic effects. Further exploration of ferroptosis will impact our understanding on how aged TME modulates immune cell killing and provide evidence to target or revert resistance to checkpoint blockade therapy associated with an IFN-y signature and new strategies for treatment of older patients. Citation Format: Murilo R. Rocha, Yash Chhabra, Laura Huser, Elizabeth Irene Harper, Agrani Dixit, Vania Wang, Alexis E. Carey, Cheyenne Palm, Fan Huang, Ashani T. Weeraratna. Ferroptosis modulates melanoma progression in an age-specific manner through changes in the tumor microenvironment [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 358.

Abstract 5513: Sexual differences in the aged melanoma tumor microenvironment dictates metastasis and therapeutic responses

Abstract There is documented sex disparity in cutaneous melanoma incidence and mortality, increasing disproportionately with age and in the male sex. However, the underlying mechanisms remain unclear; while biological sex differences and inherent immune response variability have been assessed in tumor cells, the role of the microenvironment surrounding the tumor, contextually in aging, has been overlooked. We find that skin fibroblasts undergo age-mediated changes in their proliferation, senescence, ROS levels and stress response that vary with sex. We find that aged male fibroblasts selectively drive an invasive and slow-cycling phenotype in melanoma cells in vitro by increasing AXL expression. This is also evident in syngeneic mouse models where metastasis is increased in aged male mice. Mechanistically, intrinsic aging in male fibroblasts coupled with elevated ROS promotes EZH2 decline thereby increasing BMP2 secretion, which in turn drives the slower-cycling, highly invasive, and therapy-resistant melanoma cell phenotype, characteristic of the aged male TME. Inhibition of BMP2 activity blocks the emergence of the invasive phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. Our data provides an integrated view of how age and sex of the host contributes to melanoma progression and therapy responses. Bridging this knowledge gap will improve patient stratification and assist in tailoring the therapy to the individual. Citation Format: Yash Chhabra, Mitchell Fane, Sneha Pramod, Laura Hueser, Daniel Zabransky, Vania Wang, Edwin Kumah, Alexis Carey, Elizabeth Harper, Murilo Ramos Rocha, Ashani Weeraratna. Sexual differences in the aged melanoma tumor microenvironment dictates metastasis and therapeutic responses [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 5513.

Abstract 4282: Crosstalk between the immune system and the extracellular matrix in the context of melanoma and aging

Abstract Aging is an independent prognostic factor for the diagnosis of melanoma. The extracellular matrix of the skin has well-documented changes with age, such as the loss of HAPLN1, which can lead to differential metastasis in aged individuals. The aged microenvironment can contribute to the phenotype switch, where aged individuals have smaller primary tumors but more metastases, i.e., going instead of growing. The extracellular matrix (ECM) has been shown to modulate the immune response either through direct binding of ECM proteins or through mechanotransduction, but it is unclear how aging affects this crosstalk. Our hypothesis is that the ECM can modulate immune response to melanoma and affect response to immune checkpoint blockade (ICB) therapy. Melanoma cells cultured on cell derived matrices (CDMs) from young and aged human fibroblasts show differential levels of expression of PD-L1, a ligand that binds to PD-1 on T cells to induce T cell exhaustion. Antibodies targeting PD-1/PD-L1 are one of the main ICB treatments available to melanoma. We are also investigating T cell motility on young and aged CDMs, as well as correlating T cell infiltration in murine tumors with ECM signatures as identified by second harmonic generation microscopy. Therefore, we have compiled evidence that aging affects the crosstalk between the extracellular matrix and the immune system, providing a new horizon for therapeutic improvement of response in older patients. Citation Format: Elizabeth I. Harper, Yash Chhabra, Alexis Carey, Murilo Ramos Rocha, Laura Hüser, Vania Wang, Agrani Dixit, Ashani Weeraratna. Crosstalk between the immune system and the extracellular matrix in the context of melanoma and aging [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 4282.

Abstract 6631: Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression

Abstract Advancing age is the strongest independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC) and a negative prognostic factor. PDACs are typically immunologically “cold” tumors and do not often respond to currently approved immunotherapy treatment approaches. How aging may alter non-malignant cells, such as fibroblasts and immune cell populations, in the PDAC tumor microenvironment (TME) to decrease anti-tumor immune responses is not understood. We identified that aged microenvironments significantly alter the growth and progression of PDAC tumors. Orthotopic KPC and Panc02 mouse PDAC cell line injections into the pancreata of aged (>64-week-old) compared to young (6-8-week-old) C57BL/6 mice result in more rapid tumor growth and increased frequency of liver metastases in vivo. Tumors from aged mice have high expression of the TGFβ family member GDF-15 and hyperactivation of AKT signaling driven by the release of secreted factors from aged pancreatic fibroblasts. We found that in aged microenvironments there is a marked reduction in the number of CD8+ T cells and an increase in FoxP3+ regulatory T cells in primary orthotopic pancreatic tumors using the KPC and Panc02 models. In addition, we performed portal vein injections to establish liver metastases to study the differences in immune cell infiltration in metastases from young vs. aged microenvironments. Aged mice form more and larger liver metastases compared to young mice, and metastatic lesions in aged mice have decreased CD8+ T cell infiltration when compared to young mice. Since tumors in aged microenvironments have increased AKT activation compared to young microenvironments, we next tested if AKT signaling was sufficient to alter immune cell infiltration in PDAC tumors. In young mice, treatment with recombinant GDF-15, which activates AKT signaling, reduced CD8+ T cell infiltration into tumors. Conversely, in KPC tumors grown in aged mice treated with the AKT inhibitor MK-2206, there was a trend toward increased CD8+ T cell infiltration and a significant reduction in immunosuppressive FoxP3+ Tregs. Together, these data highlight age-dependent differences in the immune infiltration in PDAC tumors. They suggest that activation of AKT signaling can drive features of immunosuppression in PDAC, providing the rationale for targeting AKT signaling to improve antitumor immune responses in PDAC. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Elizabeth M. Jaffee, Ashani T. Weeraratna, Daniel J. Zabransky. Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression [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 6631.

Abstract 2605: STX-1, a potent first-in-class ADC directed against DPP4 (CD26) as a biomarker of senescent tumor cells

Abstract Dipeptidyl Peptidase 4 (DPP4/CD26) is a membrane glycoprotein involved in particular chemotactic and inflammatory responses, via its N-terminal protein cleavage enzymatic activities. DPP4 expression is found in various cell types, in certain subsets of lymphocytes and frequently overexpressed in primary tumors and metastases where its role remains still poorly understood. Recently, DPP4 was identified as a novel biomarker of senescent cells. In cancers, the harmful effect of senescent tumor cells is well recognized and mediated by the local production of pro-inflammatory cytokines, soluble immunosuppressive factors and metalloproteinases, called ‘SASP’ phenotype which could be induced by many chemotherapy and radiation regimens. This deleterious microenvironment promotes tumor growth, invasion and neoangiogenesis which can lead to treatment resistance, relapses and poor prognosis in many cancers, especially at advanced stages. STX-1 is a leading first-in-class ADC platform based on a proprietary anti-DPP4 mAb generated after a screen between normal and senescence-induced cells identifying DPP4 as a candidate for cancer targeting. Other biomarkers of interest were also identified as being selectively expressed for further preclinical evaluation. This IgG1/k mAb has potent in vitro activities on DPP4+ cells: i) in a bleomycin-induced senescent fibroblasts model; ii) on DPP4-expressing and chemotherapy- or radiotherapy-induced senescent human tumor cell lines with overexpression of DPP4; iii) binding of the mAb to its target results in its internalization into the endolysosomal compartment of cells. The first formulations of STX-1 are undergoing preclinical evaluation in vitro and in vivo and consist of a classical maleimide-based conjugation, an enzymatically cleavable cathepsin-dipeptide linker comprising a self-immolative spacer and either inhibitor of tubulin (MMAE/DAR4) or topoisomerase I inhibitor (exatecan/DAR8) payloads. Plasma stability and affinity, dose-response potency and permeability bystander activity on human tumor cell lines were evaluated in vitro. Preliminary efficacy is demonstrated in vivo on tumor xenograft models with various DPP4 expressions (constitutive or chemo/radio-induced to senescence) based on a 21-28 days weekly infusion schedule (3 or 5 µg/kg). The indications selected for further therapeutic development are prostate, liver and colorectal carcinoma models. Data on antitumor efficacy, tolerability and pharmacokinetics will be presented. Further studies will include optimization of the best STX-1 formulation, NHP studies and GMP scale-up with planned IND submission expected in Q3-2024. Exploiting the targeting of senescent cells in cancer based on selected biomarkers such as DPP4 constitutes a new therapeutic avenue to counteract tumor dormancy, recurrences and resistance to conventional chemo/radiotherapies. Citation Format: Eric Angevin, Virginie Lelarge, Remi Capelle, Justine Choeur, Thierry Mathieu, Benjamin Le Calvé. STX-1, a potent first-in-class ADC directed against DPP4 (CD26) as a biomarker of senescent tumor cells [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 2605.

Induction of Autophagy by Extract from Corydalis heterocarpa for Skin Anti-Aging.

The extracts of Corydalis heterocarpa, a salt-tolerant plant, exhibit diverse physiological properties, including anti-inflammatory, anticancer, and antiadipogenic effects. However, the anti-aging effects of C. heterocarpa extract (CHE) on human skin cells have not yet been investigated. In the present study, we determined that CHE inhibited senescence-associated β-galactosidase (SA-β-gal)-stained senescent human dermal fibroblasts (HDFs). Furthermore, CHE markedly suppressed the expression of major regulatory proteins involved in senescence, including p53, p21, and caveolin-1. Interestingly, CHE promoted autophagic flux, as confirmed by the formation of microtubule-associated protein 1 light chain 3B (LC3B) puncta and lysosomal activity. Notably, using RNA sequencing (RNA-seq), we showed that CHE selectively regulated the gene expression of leucine-rich repeat and sterile alpha motif-containing 1 (LRSAM1), an important regulator of autophagy. The adenosine-monophosphate activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway, which is essential for autophagy regulation, was also modulated by CHE. LRSAM1 depletion not only inhibited LC3B expression but also decreased the autophagy flux induced by CHE. Moreover, the knockdown of LRSAM1 suppressed the reversal of CHE-induced senescence in old HDFs. Collectively, our study has revealed the rejuvenating effects and molecular mechanisms of CHE, suggesting that CHE may be a promising anti-aging agent.

Purinergic signaling promotes premature senescence

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.

Involvement of retinoic acid‑inducible gene‑I in radiation‑induced senescence of human umbilical vein endothelial cells.

In 2012, the threshold radiation dose (0.5 Gy) for cardiovascular and cerebrovascular diseases was revised, and this threshold dose may be exceeded during procedures involving radiation such as interventional radiology. Therefore, in addition to regulating radiation dose, it is necessary to develop strategies to prevent and mitigate the development of cardiovascular disease. Cellular senescence is irreversible arrest of cell proliferation. Although cellular senescence is one of the mechanisms for suppressing cancer, it also has adverse effects. For example, senescence of vascular endothelial cells is involved in development of vascular disorders. However, the mechanisms underlying induction of cellular senescence are not fully understood. Therefore, the present study explored the factors involved in the radiation-induced senescence in human umbilical vein endothelial cells (HUVECs). The present study reanalyzed the gene expression data of senescent normal human endothelial cells and fibroblast after irradiation (NCBI Gene Expression Omnibus accession no. GSE130727) and microarray data of HUVECs 24 h after irradiation (NCBI Gene Expression Omnibus accession no. GSE76484). Numerous genes related to viral infection and inflammation were upregulated in radiation-induced senescent cells. In addition, the gene group involved in the retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling pathway, which plays an important role to induce anti-viral response, was altered in irradiated HUVECs. Therefore, to investigate the involvement of RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are RLRs, in radiation-induced senescence of HUVECs, the protein expression of RIG-I and MDA5 and the activity of senescence-associated β-galactosidase (SA-β-gal), a representative senescence marker, were analyzed. Of note, knockdown of RIG-I in HUVECs significantly decreased radiation-increased proportion of cells with high SA-β-gal activity (i.e., senescent cells), whereas this phenomenon was not observed in MDA5-knockdown cells. Taken together, the present results suggested that RIG-I, but not MDA5, was associated with radiation-induced senescence in HUVECs.

Regulation of ENPP5, a senescence-associated secretory phenotype factor, prevents skin aging.

Aging negatively affects the appearance and texture of the skin owing to the accumulation of senescent fibroblasts within the dermis. Senescent cells undergo abnormal remodeling of collagen and the extracellular matrix through an inflammatory histolytic senescence-associated secretory phenotype (SASP). Therefore, suppression of SASP in senescent cells is essential for the development of effective skin anti-aging therapies. Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5), an extracellular signaling molecule, has been implicated in vascular aging and apoptosis; however, its role in SASP remains unclear. Therefore, this study aimed to investigate the role of ENPP5 in SASP and skin aging using molecular techniques. We investigated the effects of siRNA-mediated ENPP5 knockdown, human recombinant ENPP5 (rENPP5) treatment, and lentiviral overexpression of ENPP5 on SASP and aging in human skin fibroblasts. Additionally, we investigated the effect of siRNA-mediated ENPP5 knockdown on the skin of C57BL/6 mice. We found that ENPP5 was significantly expressed in replication-aged and otherwise DNA-damaged human skin fibroblasts and that treatment with human rENPP5 and lentiviral overexpression of ENPP5 promoted SASP and senescence. By contrast, siRNA-mediated knockdown of ENPP5 suppressed SASP and the expression of skin aging-related factors. Additionally, ENPP5 knockdown in mouse skin ameliorated the age-related reduction of subcutaneous adipose tissue, the panniculus carnosus muscle layer, and thinning of collagen fibers. Conclusively, these findings suggest that age-related changes may be prevented through the regulation of ENPP5 expression to suppress SASP in aging cells, contributing to the development of anti-aging treatments for the skin.

Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. In this study, using budding yeast and normal human fibroblasts, we found that cellular senescence—stable cell cycle arrest contributing to organismal aging—is the long-term outcome of PMD. Our genetic screening using budding yeast unexpectedly identified a close genetic association between PMD response and replicative lifespan regulations. Furthermore, PMD limits replicative lifespan in budding yeast; upregulation of membrane repair factors ESCRT-III (SNF7) and AAA-ATPase (VPS4) extends it. In normal human fibroblasts, PMD induces premature senescence via the Ca2+–p53 axis but not the major senescence pathway, DNA damage response pathway. Transient upregulation of ESCRT-III (CHMP4B) suppressed PMD-dependent senescence. Together with mRNA sequencing results, our study highlights an underappreciated but ubiquitous senescent cell subtype: PMD-dependent senescent cells.

Mapping the core senescence phenotype of primary human colon fibroblasts.

Advanced age is the largest risk factor for many diseases and several types of cancer, including colorectal cancer (CRC). Senescent cells are known to accumulate with age in various tissues, where they can modulate the surrounding tissue microenvironment through their senescence associated secretory phenotype (SASP). Recently, we showed that there is an increased number of senescent cells in the colons of CRC patients and demonstrated that senescent fibroblasts and their SASP create microniches in the colon that are conducive to CRC onset and progression. However, the composition of the SASP is heterogenous and cell-specific, and the precise senescence profile of colon fibroblasts has not been well-defined. To generate a SASP atlas of human colon fibroblasts, we induced senescence in primary human colon fibroblasts using various in vitro methods and assessed the resulting transcriptome. Using RNASequencing and further validation by quantitative RT-PCR and Luminex assays, we define and validate a 'core senescent profile' that might play a significant role in shaping the colon microenvironment. We also performed KEGG analysis and GO analyses to identify key pathways and biological processes that are differentially regulated in colon fibroblast senescence. These studies provide insights into potential driver proteins involved in senescence-associated diseases, like CRC, which may lead to therapies to improve overall health in the elderly and to prevent CRC.

Senescent fibroblast facilitates re-epithelization and collagen deposition in radiation-induced skin injury through IL-33-mediated macrophage polarization

BackgroundThe need for radiotherapy among the elderly rises with increasing life expectancy and a corresponding increase of elderly cancer patients. Radiation-induced skin injury is one of the most frequent adverse effects in radiotherapy patients, severely limiting their life quality. Re-epithelialization and collagen deposition have essential roles in the recovery of skin injuries induced by high doses of ionizing radiation. At the same time, radiation-induced senescent cells accumulate in irradiated tissues. However, the effects and mechanisms of senescent cells on re-epithelialization and collagen deposition in radiation-induced skin injury have not been fully elucidated.ResultsHere, we identified a role for a population of senescent cells expressing p16 in promoting re-epithelialization and collagen deposition in radiation-induced skin injury. Targeted ablation of p16+ senescent cells or treatment with Senolytics resulted in the disruption of collagen structure and the retardation of epidermal coverage. By analyzing a publicly available single-cell sequencing dataset, we identified fibroblasts as a major contributor to the promotion of re-epithelialization and collagen deposition in senescent cells. Notably, our analysis of publicly available transcriptome sequencing data highlighted IL-33 as a key senescence-associated secretory phenotype produced by senescent fibroblasts. Neutralizing IL-33 significantly impedes the healing process. Finally, we found that the effect of IL-33 was partly due to the modulation of macrophage polarization.ConclusionsIn conclusion, our data suggested that senescent fibroblasts accumulated in radiation-induced skin injury sites participated in wound healing mainly by secreting IL-33. This secretion regulated the local immune microenvironment and macrophage polarization, thus emphasizing the importance of precise regulation of senescent cells in a phased manner.Graphical

Identification of the role of TG2 on the expression of TGF-β, TIMP-1 and TIMP-2 in aged skin

Abstract Objectives Transglutaminase 2 (TG2) is a unique protein having enzymatic and nonenzymatic functions that have been implicated in various biological and pathological processes such as cell survival and apoptosis, cell signaling, differentiation, adhesion and migration, wound healing and inflammation. As reported in previous studies, TG2 expression and activity increase by age suggesting that TG2 possibly has roles in cellular aging process. In this study, we aimed to explore the role of TG2 in chronological skin aging through its impact on the expression of some important extracellular matrix (ECM) proteins including TGF-β, TIMP-1 and TIMP-2. Methods We have compared TG2 expression and activity in young and in vitro chronologically aged human dermal fibroblasts via Western blot and in situ TG2 activity assays. Afterwards, we inhibited TG2 expression via siRNA transfection and activity via active site inhibitor of TG2 separately in aged dermal fibroblasts and monitored the expression levels of TGF-β, TIMP-1 and TIMP-2 in these cells by Western blot and compared to that of untreated control cells. Results We obtained evidence that both TG2 expression and activity increase in aged cells. However, protein levels of TGF-β, TIMP-1 and TIMP-2 do not exhibit any significant difference in TG2 downregulated or TG2 activity inhibited aged cells compared to control cells. Conclusions Our results indicate that changes in the expression and activity of TG2 in (in vitro) chronologically aged human dermal fibroblasts do not impact the expression patterns of TGF-β, TIMP-1 and TIMP-2 proteins.

Effect of Mexican Propolis on Wound Healing in a Murine Model of Diabetes Mellitus.

Diabetes mellitus (DM) affects the wound healing process, resulting in impaired healing or aberrant scarring. DM increases reactive oxygen species (ROS) production, fibroblast senescence and angiogenesis abnormalities, causing exacerbated inflammation accompanied by low levels of TGF-β and an increase in Matrix metalloproteinases (MMPs). Propolis has been proposed as a healing alternative for diabetic patients because it has antimicrobial, anti-inflammatory, antioxidant and proliferative effects and important properties in the healing process. An ethanolic extract of Chihuahua propolis (ChEEP) was obtained and fractionated, and the fractions were subjected to High-Performance Liquid Chromatography with diode-array (HPLC-DAD), High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) analyses and 46 compounds were detected. Deep wounds were made in a murine DM model induced by streptozotocin, and the speed of closure and the wound tensile strength were evaluated by the tensiometric method, which showed that ChEEP had similar activity to Recoveron, improving the speed of healing and increasing the wound tensile strength needed to open the wound again. A histological analysis of the wounds was performed using H&E staining, and when Matrix metalloproteinase 9 (MMP9) and α-actin were quantified by immunohistochemistry, ChEEP was shown to be associated with improved histological healing, as indicated by the reduced MMP9 and α-actin expression. In conclusion, topical ChEEP application enhances wound healing in diabetic mice.

Highly Active Frozen Nanovesicles Microneedles for Senile Wound Healing via Antibacteria, Immunotherapy, and Skin Regeneration.

Senile wound healing risks a variety of health complications and makes both economic and psychological burdens on patients greatly. Poor activity of aged dermal fibroblasts (A-FBs) and local disordered immunoreaction in the deep dermis contribute to delayed wound healing. Therefore, the locally complex microenvironment in deep requires additional processing. Herein, a novel double-layer hyaluronic acid methacrylate (HAMA)/polyvinyl alcohol (PVA) microneedle patch (MNP) coated by young fibroblast-derived exosomes (Y-EXOs) (Y-EXOs@HAMA/PVA MNP) is presented for deep drug delivery, aged wound healing and immunoregulation. A spraying and freeze-drying method is applied for keeping the bioactivity of the nanovesicles. An ideal loading of Y-EXOs and enhanced strength for penetration have realized after circulation for times. The Y-EXOs@HAMA/PVA MNP shows an excellent influence on delayed wound healing of aged skin with active A-FBs, more deposition of collagen and less production of IL-17A compared with application of aged fibroblast-derived exosomes (A-EXOs). Moreover, the content microRNAs in Y-EXOs and A-EXOs are sequenced for further study. This study initiatively demonstrates that Y-EXOs have effective function on both anti-aging and anti-inflammation and Y-EXOs@HAMA/PVA MNP is expected as a novel strategy for deep drug delivery for promoting hard wound healing in aged skin in future clinical application.