Senescence Secretome Research Articles

Molecular Signatures of Senescence in Periodontitis: Clinical Insights.

Most of the elderly population is afflicted by periodontal diseases, creating a health burden worldwide. Cellular senescence is one of the hallmarks of aging and associated with several chronic comorbidities. Senescent cells produce a variety of deleterious secretions, collectively termed the senescence-associated secretory phenotype (SASP). This disrupts neighboring cells, leading to further senescence propagation and inciting chronic inflammation, known as "inflammaging." Detrimental repercussions within the tissue microenvironment can trigger senescence at a younger age, accelerate biological aging, and drive the initiation or progression of diseases. Here, we investigated the biological signatures of senescence in healthy and diseased gingival tissues by assessing the levels of key senescence markers (p16, lipofuscin, and β-galactosidase) and inflammatory mediators (interleukin [IL]-1β, IL-6, IL-8, matrix metalloproteinase [MMP]-1, MMP-3, and tumor necrosis factor-α). Our results showed significantly increased senescence features including p16, lipofuscin, and β-galactosidase in both epithelial and connective tissues of periodontitis patients compared with healthy sites in all age groups, indicating that an inflammatory microenvironment can trigger senescence-like alterations in younger diseased gingival tissues as well. Subsequent analyses using double staining with specific cell markers noted the enrichment of β-galactosidase in fibroblasts and macrophages. Concurrently, inflammatory mediators consistent with SASP were increased in the gingival biopsies obtained from periodontitis lesions. Together, our findings provide the first clinical report revealing susceptibility to elevated senescence and inflammatory milieu consistent with senescence secretome in gingival tissues, thus introducing senescence as one of the drivers of pathological events in the oral mucosa and a novel strategy for targeted interventions.

Senescent response in inner annulus fibrosus cells in response to TNFα, H2O2, and TNFα-induced nucleus pulposus senescent secretome.

Senescence, particularly in the nucleus pulposus (NP) cells, has been implicated in the pathogenesis of disc degeneration, however, the mechanism(s) of annulus fibrosus (AF) cell senescence is still not well understood. Both TNFα and H2O2, have been implicated as contributors to the senescence pathways, and their levels are increased in degenerated discs when compared to healthy discs. Thus, the objective of this study is to identify factor(s) that induces inner AF (iAF) cell senescence. Under TNFα exposure, at a concentration previously shown to induce senescence in NP cells, bovine iAF cells did not undergo senescence, indicated by their ability to continue to proliferate as demonstrated by Ki67 staining and growth curves and lack of expression of the senescent markers, p16 and p21. The lack of senescent response occurred even though iAF express higher levels of TNFR1 than NP cells. Interestingly, iAF cells showed no increase in intracellular ROS or secreted H2O2 in response to TNFα which contrasted to NP cells that did. Following TNFα treatment, only iAF cells had increased expression of the superoxide scavengers SOD1 and SOD2 whereas NP cells had increased NOX4 gene expression, an enzyme that can generate H2O2. Treating iAF cells with low dose H2O2 (50 μM) induced senescence, however unlike TNFα, H2O2 did not induce degenerative-like changes as there was no difference in COL2, ACAN, MMP13, or IL6 gene expression or number of COL2 and ACAN immunopositive cells compared to untreated controls. The latter result suggests that iAF cells may have distinct degenerative and senescent phenotypes. To evaluate paracrine signalling by senescent NP cells, iAF and TNFα-treated NP cells were co-cultured. In contact co-culture the NP cells induced iAF senescence. Thus, senescent NP cells may secrete soluble factors that induce degenerative and senescent changes within the iAF. This may contribute to a positive feedback loop of disc degeneration. It is possible these factors may include H2O2 and cytokines (such as TNFα). Further studies will investigate if human disc cells respond similarly.

A navitoclax-loaded nanodevice targeting matrix metalloproteinase-3 for the selective elimination of senescent cells

Cellular senescence is implicated in the occurrence and progression of multiple age-related disorders. In this context, the selective elimination of senescent cells, senolysis, has emerged as an effective therapeutic strategy. However, the heterogeneous senescent phenotype hinders the discovery of a universal and robust senescence biomarker that limits the effective of senolytic with off-target toxic effects. Therefore, the development of more selective strategies represents a promising approach to increase the specificity of senolytic therapy. In this study, we have developed an innovative nanodevice for the selective elimination of senescent cells (SCs) based on the specific enzymatic activity of the senescent secretome. The results revealed that when senescence is induced in proliferating WI-38 by ionizing radiation (IR), the cells secrete high levels of matrix metalloproteinase-3 (MMP-3). Based on this result, mesoporous silica nanoparticles (MSNs) were loaded with the senolytic navitoclax (Nav) and coated with a specific peptide which is substrate of MMP-3 (NPs(Nav)@MMP-3). Studies in cells confirmed the preferential release of cargo in IR-induced senescent cells compared to proliferating cells, depending on MMP-3 levels. Moreover, treatment with NPs(Nav)@MMP-3 induced a selective decrease in the viability of SCs as well as a protective effect on non-proliferating cells. These results demonstrate the potential use of NPs to develop enhanced senolytic therapies based on specific enzymatic activity in the senescent microenvironment, with potential clinical relevance. Statement of significanceThe common β-galactosidase activity has been exploited to develop nanoparticles for the selective elimination of senescent cells. However, the identification of new senescent biomarkers is a key factor for the development of improved strategies. In this scenario, we report for the first time the development of NPs targeting senescent cells based on specific enzymatic activity of the senescent secretome. We report a navitoclax-loaded nanodevice responsive to the matrix metalloproteinase-3 (MMP-3) associated with the senescent phenotype. Our nanosystem achieves the selective release of navitoclax in an MMP-3-dependent manner while limiting off-target effects on non-senescent cells. This opens the possibility of using nanoparticles able to detect an altered senescent environment and selectively release its content, thus enhancing the efficacy of senolytic therapies.

The senescent secretome drives PLVAP expression in cultured human hepatic endothelial cells to promote monocyte transmigration

Liver sinusoidal endothelial cells (LSEC) undergo significant phenotypic change in chronic liver disease (CLD), and yet the factors that drive this process and the impact on their function as a vascular barrier and gatekeeper for immune cell recruitment are poorly understood. Plasmalemma-vesicle-associated protein (PLVAP) has been characterized as a marker of LSEC in CLD; notably we found that PLVAP upregulation strongly correlated with markers of tissue senescence. Furthermore, exposure of human LSEC to the senescence-associated secretory phenotype (SASP) led to a significant upregulation of PLVAP. Flow-based assays demonstrated that SASP-driven leukocyte recruitment was characterized by paracellular transmigration of monocytes while the majority of lymphocytes migrated transcellularly. Knockdown studies confirmed that PLVAP selectively supported monocyte transmigration mediated through PLVAP's impact on LSEC permeability by regulating phospho-VE-cadherin expression and endothelial gap formation. PLVAP may therefore represent an endothelial target that selectively shapes the senescence-mediated immune microenvironment in liver disease.

Exactis-03: A phase I trial of the combination of olaparib and navitoclax in women with high grade serous ovarian cancer and triple negative breast cancer.

TPS5623 Background: Identifying new therapeutic options for patients (pts) with high grade serous ovarian cancer (HGSC) and triple negative breast cancer (TNBC) is a priority. For pts with pathogenic variants in BRCA, the PARP inhibitor (PARPi) olaparib is used to treat TNBC and as maintenance for HGSC. PARPi can induce a senescence-like phenotype in cancer cells with cell survival dependent on anti-apoptotic proteins. The intrinsic apoptotic pathway is regulated by the Bcl-2 family composed of anti-apoptotic proteins (including Bcl-2, Bcl-xL), pro-apoptotic effectors and the pro-apoptotic BH3-only proteins. Increased expression of Bcl-xL occurs in 88% of HGSC. Olaparib and navitoclax, a Bcl-2/Bcl-xL inhibitor, are synergistic in pre clinical models of HGSC and TNBC. Exactis- 03 investigates targeting, olaparib-induced sensecence as a treatment strategy for pts with HGSC and TNBC. Methods: Exactis-03 is a multi-centre phase I trial determining if olaparib can be safely combined with navitoclax in pts with TNBC who have somatic or germline mutations in BRCA1/2 or PALB2 and pts with recurrent HGSC who have progressed ≥ 6 months since their last platinum based chemotherapy. Additional eligibility criteria include : ≥3 prior lines of treatment for TNBC (no limit for HGSC); for pts with HGSC prior PARPi is allowed provided there was no progression on or ≤6 months since discontinuation of the PARPi; ECOG PS ≤2; ability to absorb study medication and pts must be willing and able to undergo study related procedures. The primary endpoint is identification of the recommended phase II dose (RP2D) of olaparib combined with navitoclax. Olaparib 200mg bid is administered alone for the first 2 weeks. Tumor biopsies will be performed at baseline and on day 7-12 (prior to navitoclax) to evaluate senescence and apoptosis biomarkers and to create 3D organoids and ex vivo microdissected tumor (MDT) models for functional assessment of drug response. Navitoclax is dose escalated with a fixed dose of olaparib in 28-day cycles with 3 pts initially treated at each dose level. If no dose limiting toxicity (DLT) is observed, the dose level will be escalated until ≥ 1/3 or ≥ 2/6 patients experience DLT. The RP2D will be defined as the dose level below the one where ≥ 1/3 or ≥ 2/6 patients experience DLT. The 2-week lead-in with olaparib alone and one full cycle of the combination (navitoclax/olaparib) must be completed before dose escalation is permitted. Blood samples are collected for pharmacokinetics and serial evaluation of plasma biomarkers. There is an optional tumor biopsy on progression. Exploratory objectives include determining levels of pro- and anti- apoptotic proteins in biopsy samples and evaluation of cell fate decision biomarkers in biopsy tissue and blood (senescence secretome). Patient-derived organoids and ex vivo MDT will be used to explore drug response and sequencing (NCT05358639). Clinical trial information: NCT05358639 .

Abstract 5504: A tumor-promoting senescent secretome triggered by platinum chemotherapy exploits a targetable TGFβR1/Akt-mTOR axis in lung cancer

Abstract Background Platinum-based chemotherapy is commonly used for the treatment of non-small cell lung cancer (NSCLC), yet clinical outcomes and survival rates remain very poor and continues to be a cancer of unmet need. Recent evidence points to cellular senescence, a response to oncogenic- and therapy-induced genotoxic stress, and its associated proinflammatroy secretory phenotype (SASP) as an emerging hallmark of cancer. Hence, targeting senescence and its associated tumor-promoting activities is emerging as a novel and promising therapeutic strategy but largely unexplored in lung cancer. Experimental Procedures Our study includes a variety of methodologies, including: - Functional in vitro analyses: proliferation, colony formation, tumor spheres, migration assays. High throughput unbiased analyses: RNAseq, proteomics and microenvironment microarrays (MEMA). - In vivo models of lung cancer: xenografts, orthotopic and genetically engineered mouse models. Longitudinal tumor burden by IVIS and microCT and mouse survival. - Clinical samples: Histological and in silico analyses. Results Here we show that cisplatin-derived SASP enhances the malignant phenotype of lung cancer cells. Using xenograft, orthotopic and KrasG12V-driven murine NSCLC models, we demonstrate that cisplatin-induced senescent cells strongly promote tumor progression. Mechanistically, we find that a TGF-β-enriched SASP drives pro-proliferative effects through TGFβR1 and Akt/mTOR pathway activation. We validate the translational relevance of chemotherapy-induced SASP using clinical NSCLC samples from a trial with patients who received neoadjuvant platinum-based chemotherapy. Importantly, TGFβR1 inhibition with galunisertib or senolytic treatment significantly reduces tumor promotion driven by cisplatin-induced senescence. Finally, we demonstrate, using distinct murine NSCLC models, that addition of TGFBR1 inhibitors to platinum-based chemotherapy reduces tumor burden and improves survival, providing pre-clinical proof-of-concept for future trial designs on combination therapies. Conclusions and Impact We regard this work as a major conceptual dissection of tumor-promoting activities of therapy-induced senescence, and a preclinical advance in the management of lung cancer with potential wide therapeutic applications in precision medicine. Of note, we expect our findings to have implications for multiple cancer types, including ovarian, breast, mesothelioma, oesophageal, head and neck, bladder and brain cancers, where platinum-based therapies remain important standard-of-care treatments. Citation Format: Estela González-Gualda, David Macias, Samir Morsli, José Ezequiel Martín, Hui-Ling Ou, Mary Denholm, Ioana Olan, Reuben Hoffmann, Mark Dane, Dimitris Veroutis, Guillermo Medrano, Francisca Mulero, Carla P. Martins, Mariano Barbacid, Vassilis Gorgoulis, James E. Korkola, Doris M. Rassl, Gary J. Doherty, Robert C. Rintoul, Masashi Narita, Daniel Muñoz-Espín. A tumor-promoting senescent secretome triggered by platinum chemotherapy exploits a targetable TGFβR1/Akt-mTOR axis in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5504.

Insulin-like growth factor binding protein 7 (IGFBP7), a link between heart failure and senescence.

Insulin like growth factor binding protein 7 (IGFBP7) is a marker of senescence secretome and a novel biomarker in patients with heart failure (HF). We evaluated the prognostic value of IGFBP7 in patients with heart failure and examined associations to uncover potential new pathophysiological pathways related to increased plasma IGFBP7 concentrations. We have measured plasma IGFBP7 concentrations in 2250 subjects with new-onset or worsening heart failure (BIOSTAT-CHF cohort). Higher IGFBP7 plasma concentrations were found in older subjects, those with worse kidney function, history of atrial fibrillation, and diabetes mellitus type 2, and in subjects with higher number of HF hospitalizations. Higher IGFBP7 levels also correlate with the levels of several circulating biomarkers, including higher NT-proBNP, hsTnT, and urea levels. Cox regression analyses showed that higher plasma IGFBP7 concentrations were strongly associated with increased risk of all three main endpoints (hospitalization, all-cause mortality, and combined hospitalization and mortality) (HR 1.75, 95% CI 1.25-2.46; HR 1.71, 95% CI 1.39-2.11; and HR 1.44, 95% CI 1.23-1.70, respectively). IGFBP7 remained a significant predictor of these endpoints in patients with both reduced and preserved ejection fraction. Likelihood ratio test showed significant improvement of all three risk prediction models, after adding IGFBP7 (P<0.001). A biomarker network analysis showed that IGFBP7 levels activate different pathways involved in the regulation of the immune system. Results were externally validated in BIOSTAT-CHF validation cohort. IGFPB7 presents as an independent and robust prognostic biomarker in patients with HF, with both reduced and preserved ejection fraction. We validate the previously published data showing IGFBP7 has correlations with a number of echocardiographic markers. Lastly, IGFBP7 pathways are involved in different stages of immune system regulation, linking heart failure to senescence pathways.

Synergism of BCL-2 family inhibitors facilitates selective elimination of senescent cells

Accumulation of senescent cells in tissues with advancing age participates in the pathogenesis of several human age-associated diseases. Specific senescent secretome, the resistance of senescent cells to apoptotic stimuli, and lack of immune system response contribute to the accumulation of senescent cells and their adverse effects in tissues. Inhibition of antiapoptotic machinery, augmented in senescent cells, by BCL-2 protein family inhibitors represents a promising approach to eliminate senescent cells from tissues. This study aimed to explore synergistic and selective senolytic effects of anti-apoptotic BCL-2 family targeting compounds, particularly BH3 mimetics.Using human non-transformed cells RPE-1, BJ, and MRC-5 brought to ionizing radiation-, oncogene-, drug-induced and replicative senescence, we found synergy in combining MCL-1 selective inhibitors with other BH3 mimetics. In an attempt to uncover the mechanism of such synergy, we revealed that the surviving subpopulation of cells resistant to individually applied ABT-737/ABT-263, MIK665, ABT-199, and S63845 BCL-2 family inhibitors showed elevated MCL-1 compared to untreated control cells indicating the presence of a subset of cells expressing high MCL-1 levels and, therefore, resistant to BCL-2 inhibitors within the original population of senescent cells.Overall, we found that combining BCL-2 inhibitors can be beneficial for eliminating senescent cells, thereby enabling use of lower, potentially less toxic, doses of drugs compared to monotherapy, thereby overcoming the resistance of the subpopulation of senescent cells to monotherapy.

Secretome of senescent hepatoma cells modulate immune cell fate by macrophage polarization and neutrophil extracellular traps formation

Presence of dysfunctional senescent hepatocytes is a hallmark feature of liver cirrhosis which finally culminates in liver cancer. We now report the presence of senescent hepatocytes (p21 and p53 positive) in the vicinity of infiltrated immune cells in hepatocellular carcinoma tissue specimens by immunohistochemistry. Hence, we evaluated in vitro, the relevance of senescent hepatoma cells in altering the fate of monocytes and neutrophils by assaying for macrophage polarization and extracellular trap (NETs) formation, respectively. Premature senescence was induced in hepatoma cells (HepG2 and Huh7 cells) by treating cells with doxorubicin. Senescent hepatoma cells showed strong inflammatory phenotype with induced expression of cytokines (IL1β, IL6, IL8 and IL13) as evaluated by flow cytometry. The senescent secretome from hepatoma cells when incubated with healthy monocytes caused it to differentiate predominantly towards M2 fate (CD80low CD86low CD163high CD206high) when analysed by flow cytometry. This was corroborated by the finding in clinical samples where human hepatocellular carcinoma harbouring senescent hepatocytes showed presence of M2 macrophages, while M1 macrophages were predominant in non-tumorous region. Additionally, the senescent secretome from Huh7 cells enhanced the NETs formation, while HepG2 secretome had an inhibitory effect. In conclusion, the "pro-inflammatory" senescent secretome drives non-inflammatory type M2 macrophage polarization and modulated neutrophil traps which in turn can influence the tumor microenvironment.

COX2 regulates senescence secretome composition and senescence surveillance through PGE2

SummarySenescent cells trigger their own immune-mediated destruction, termed senescence surveillance. This is dependent on the inflammatory senescence-associated secretory phenotype (SASP), which includes COX2, an enzyme with complex roles in cancer. The role COX2 plays during senescence surveillance is unknown. Here, we show that during RAS-induced senescence (RIS), COX2 is a critical regulator of SASP composition and senescence surveillance in vivo. COX2 regulates the expression of multiple inflammatory SASP components through an autocrine feedback loop involving its downstream product, prostaglandin E2 (PGE2), binding to EP4. During in vivo hepatocyte RIS, Cox2 is critical to tumor suppression, Cxcl1 expression, and immune-mediated senescence surveillance, partially through PGE2. Loss of Cox2 in RIS dysregulates the intrahepatic immune microenvironment, with enrichment of immunosuppressive immature myeloid cells and CD4+ regulatory T lymphocytes. Therefore, COX2 and PGE2 play a critical role in senescence, shaping SASP composition, promoting senescence surveillance and tumor suppression in the earliest stages of tumorigenesis.

Abstract PS19-13: Palmitate exacerbates breast tumorigenesis in vitro via induction of a senescent-like phenotype in fibroblasts

Abstract Background: Obesity is associated with a worse breast cancer prognosis, conferring an increased risk of recurrence and mortality. At the same time, recent evidence suggests that obesity is also correlated with development of cellular senescence, an inflammatory state associated with exacerbation of breast tumorigenesis in preclinical models. As obese individuals present greater levels of inflammation at baseline, research efforts are warranted to examine the means by which obesity promotes the development of a senescent phenotype, which may further exacerbate inflammation. Additionally, studies have yet to determine whether obesity-induced senescence modulates the tumorigenic process in the context of breast cancer specifically. Methods: To this end, we exposed fibroblasts to the obesity-associated circulating factor palmitate and used qPCR and western immunoblotting to assess the expression of genes and proteins involved in the senescent state, including interleukin (IL)-1a, IL-6, IL-8, senescence-associated beta-galactosidase (SA-beta-gal), and matrixmetalloproteinase (MMP)-9. As a mechanistic investigation, we next assessed the impact of palmitate on activation of NF-kB signaling using western immunoblotting, immunofluorescence, and NF-kB luciferase reporter assays. Finally, we utilized cell counting, MTT, wound healing, and colony formation assays to examine the impact of these palmitate-exposed fibroblasts on breast cancer cell proliferation, viability, motility, and survival, respectively. Results: We found that palmitate induced fibroblast gene expression of IL-1a, IL-6, and IL-8, major components of the senescent secretome, as well as expression of the senescent markers SA-beta-gal and MMP-9. The mechanism at least partially involved activation of NF-kB, responsible for production of about 75% of senescent secretome components. More importantly, these palmitate-exposed fibroblasts were of pathological impact, exacerbating in vitro measures of breast cancer cell aggressiveness. Conclusions: These findings contribute to our understanding of the impact of obesity-associated factors on breast tumorigenesis, demonstrating a mechanistic link between palmitate and the pro-tumorigenic effects of senescent cells. Our studies will ultimately aid in the identification of a therapeutic target that can be used to improve the comparably worse outcomes of the obese breast cancer patient. Citation Format: Brittany Susanne Harlow, Albert Davalos, Andrew Brenner, Christopher Jolly, Stefano Tiziani, Steve Hursting, Linda deGraffenried. Palmitate exacerbates breast tumorigenesis in vitro via induction of a senescent-like phenotype in fibroblasts [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-13.

Isolation methodology is essential to the evaluation of the extracellular vesicle component of the senescence-associated secretory phenotype.

A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediators and tissue remodelling agents – the senescence‐associated secretory phenotype (SASP). Through the SASP, senescent cells are hypothesised to contribute to both ageing and pathologies associated with age. Whilst soluble factors have been the most widely investigated components of the SASP, there is growing evidence that small extracellular vesicles (EVs) comprise functionally important constituents. Thus, dissecting the contribution of the soluble SASP from the vesicular component is crucial to elucidating the functional significance of senescent cell derived EVs. Here, we take advantage of a systematic proteomics based approach to determine that soluble SASP factors co‐isolate with EVs following differential ultracentrifugation (dUC). We present size‐exclusion chromatography (SEC) as a method for separation of the soluble and vesicular components of the senescent secretome and thus EV purification. Furthermore, we demonstrate that SEC EVs isolated from senescent cells contribute to non‐cell autonomous paracrine senescence. Therefore, this work emphasises the requirement for methodological rigor due to the propensity of SASP components to co‐isolate during dUC and provides a framework for future investigations of the vesicular component of the SASP.

Unique signatures of stress-induced senescent human astrocytes

Senescence was recently linked to neurodegeneration and astrocytes are one of the major cell types to turn senescent under neurodegenerative conditions. Senescent astrocytes were detected in Parkinson's disease (PD) patients' brains besides reactive astrocytes, yet the difference between senescent and reactive astrocytes is unclear. We aimed to characterize senescent astrocytes in comparison to reactive astrocytes and investigate differences and similarities. In a cell culture model of human fetal astrocytes, we determined a unique senescent transcriptome distinct from reactive astrocytes, which comprises dysregulated pathways. Both, senescent and reactive human astrocytes activated a proinflammatory pattern. Astrocyte senescence was at least partially depending on active mechanistic-target-of-rapamycin (mTOR) and DNA-damage response signaling, both drivers of senescence. To further investigate how PD and senescence connect to each other, we asked if a PD-linked environmental factor induces senescence and if senescence impairs midbrain neurons. We could show that the PD-linked pesticide rotenone causes astrocyte senescence. We further delineate, that the senescent secretome exaggerates rotenone-induced neurodegeneration in midbrain neurons differentiated from human induced pluripotent stem cells (hiPSC) of PD patients with alpha-synuclein gene (SNCA) locus duplication.

FBP1 loss disrupts liver metabolism and promotes tumorigenesis through a hepatic stellate cell senescence secretome.

Crosstalk between deregulated hepatocyte metabolism and cells within the tumour microenvironment, and consequent effects on liver tumourigenesis, are incompletely understood. We show here that hepatocyte-specific loss of the gluconeogenic enzyme fructose 1,6-bisphosphatase 1 (FBP1) disrupts liver metabolic homeostasis and promotes tumour progression. FBP1 is universally silenced in both human and murine liver tumours. Hepatocyte-specific Fbp1 deletion results in steatosis, concomitant with activation and senescence of hepatic stellate cells (HSCs), exhibiting a senescence-associated secretory phenotype (SASP). Depleting senescent HSCs by “senolytic” treatment with dasatinib/quercetin or ABT-263 inhibits tumour progression. We further demonstrate that FBP1-deficient hepatocytes promote HSC activation by releasing HMGB1; blocking its release with the small molecule inflachromene limits FBP1-dependent HSC activation, subsequent SASP development, and tumour progression. Collectively, these findings provide genetic evidence for FBP1 as a metabolic tumour suppressor in liver cancer and establish a critical crosstalk between hepatocyte metabolism and HSC senescence that promotes tumour growth.

Abstract P1-08-02: Omega-3 fatty acids modulate the ability of obese conditions to induce of a proinflammatory phenotype in fibroblasts

Abstract Background: Approximately 40% of American women suffer from obesity, and about 1 in 8 women will be diagnosed with breast cancer during her lifetime. The concurrence of these trends has the potential to be particularly detrimental, as obesity confers a worse prognosis for both pre- and postmenopausal breast cancer patients. While the precise mechanisms by which obesity impacts breast cancer prognosis remain to be discovered, evidence suggests that obesity upregulates components of cancer-associated fibroblast (CAF) and senescent cell secretomes, both causally associated with carcinogenesis. Specifically, obesity has been shown to induce expression of the secretory products prostaglandin E2, high mobility group protein B1, interleukin (IL)-6, IL-1 beta, and matrix metalloproteinase-1, the expression of which can be modulated by omega-3 fatty acid-induced signaling. However, studies have yet to determine whether obesity imparts these proinflammatory phenotypes in any individual cell type in the breast tumor microenvironment as well as whether these phenotypes can be modulated by administration of omega-3 fatty acids. This said, we hypothesize that fatty acids modulate the ability of obese conditions to induce a proinflammatory senescent- or CAF-like fibroblast phenotype and thus impact breast cancer progression. Because fibroblasts constitute 80% of the tumor stromal mass, it is of the utmost importance to study obesity-stimulated changes in this cellular compartment and their effects on tumor progression. Methods: Fibroblasts were exposed to sera derived from lean or obese women with and without omega-3 fatty acid supplementation and assessed for changes in expression of proinflammatory genes, including IL-1α, IL-6, and IL-8, as well as nuclear localization of p65, a subunit of the NF-kB transcription factor, which transcribes about 75% of genes related to the senescent secretome. Conditioned media (CM) from these fibroblasts were then applied to breast cancer cells to assess measures of cancer cell aggressiveness. Finally, as an ex vivo study, breast tissue samples from lean and obese women and mice were compared for concentration of proinflammatory, senescent fibroblasts by immunohistochemical analyses. Results: Gene and protein expression analyses demonstrated that obese conditions induced a proinflammatory phenotype in fibroblasts, an effect at least partially modulated by omega-3 fatty acids. These phenotypic changes were not without pathological consequence: CM from obesity-stimulated fibroblasts impacted in vitro measures of breast cancer cell aggressiveness to a greater degree than lean sera-stimulated fibroblasts. Conclusions: While correlative, these data contribute to the identification of a link between obesity and proinflammatory, senescent phenotypes and will ultimately allow for elucidation of the means by which obese conditions confer a worse prognosis for breast cancer patients. In addition, these findings will contribute to our understanding of crosstalk within the tumor microenvironment and inform our pursuit for the development of novel therapeutic targets. Citation Format: Brittany Harlow, Albert Davalos, Andrew Brenner, Christopher Jolly, Stefano Tiziani, Stephen Hursting, Linda deGraffenried. Omega-3 fatty acids modulate the ability of obese conditions to induce of a proinflammatory phenotype in fibroblasts [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-08-02.

Elucidating Proteoform Dynamics Underlying the Senescence Associated Secretory Phenotype.

Primary diploid cells exit the cell cycle in response to exogenous stress or oncogene activation through a process known as cellular senescence. This cell-autonomous tumor-suppressive mechanism is also a major mechanism operative in organismal aging. To date, temporal aspects of senescence remain understudied. Therefore, we use quantitative proteomics to investigate changes following forced HRASG12V expression and induction of senescence across 1 week in normal diploid fibroblasts. We demonstrate that global intracellular proteomic changes correlate with the emergence of the senescence-associated secretory phenotype and the switch to robust cell cycle exit. The senescence secretome reinforces cell cycle exit, yet is largely detrimental to tissue homeostasis. Previous studies of secretomes rely on ELISA, bottom-up proteomics or RNA-seq. To date, no study to date has examined the proteoform complexity of secretomes to elucidate isoform-specific, post-translational modifications or regulated cleavage of signal peptides. Therefore, we use a quantitative top-down proteomics approach to define the molecular complexity of secreted proteins <30 kDa. We identify multiple forms of immune regulators with known activities and affinities such as distinct forms of interleukin-8, as well as GROα and HMGA1, and temporally resolve secreted proteoform dynamics. Together, our work demonstrates the complexity of the secretome past individual protein accessions and provides motivation for further proteoform-resolved measurements of the secretome.

Senolytics and Senostatics: A Two-Pronged Approach to Target Cellular Senescence for Delaying Aging and Age-Related Diseases.

Aging is the most important single risk factor for many chronic diseases such as cancer, metabolic syndrome, and neurodegenerative disorders. Targeting aging itself might, therefore, be a better strategy than targeting each chronic disease individually for enhancing human health. Although much should be achieved for completely understanding the biological basis of aging, cellular senescence is now believed to mainly contribute to organismal aging via two independent, yet not mutually exclusive mechanisms: on the one hand, senescence of stem cells leads to exhaustion of stem cells and thus decreases tissue regeneration. On the other hand, senescent cells secrete many proinflammatory cytokines, chemokines, growth factors, and proteases, collectively termed as the senescence-associated secretory phenotype (SASP), which causes chronic inflammation and tissue dysfunction. Much effort has been recently made to therapeutically target detrimental effects of cellular senescence including selectively eliminating senescent cells (senolytics) and modulating a proinflammatory senescent secretome (senostatics). Here, we discuss current progress and limitations in understanding molecular mechanisms of senolytics and senostatics and therapeutic strategies for applying them. Furthermore, we propose how these novel interventions for aging treatment could be improved, based on lessons learned from cancer treatment.

The senescence-associated secretory phenotype (SASP) from mesenchymal stromal cells impairs growth of immortalized prostate cells but has no effect on metastatic prostatic cancer cells.

Senescent cells secrete inflammatory cytokines, proteases, and other factors, which are indicated as senescence-associated secretory phenotype (SASP). There are contrasting studies on the role of the SASP in cancer. Studies suggested that cancer cells may misuse the senescent secretome for their growth. Other investigations evidenced that the SASP may induce cancer growth arrest, senescence, or apoptosis. These conflicting data can be reconciled considering that cancer cells can coax senescent cells to secrete factors for their survival, thus abrogating the SASP’s anti-cancer effect. Cancer stage may also have an impact on the capacity of the SASP to block tumor proliferation and promote senescence. Indeed, senescence is associated with a permanent cell cycle arrest, which needs functional cell cycle checkpoints. We evaluated the SASP effect on the in vitro biological properties of PNT2 and PC3 cells, which are immortalized prostate cells and metastatic prostatic cancer cells, respectively. We evidenced that SASPs, coming either from mesenchymal stromal cells treated with H202 or with low X-ray doses, induced senescence of immortalized cells but not of cancer cells. Hence, the SASP released by acute senescent cells should be considered as an effective weapon against pre-tumorigenesis events rather than an anti-cancer mechanism acting on malignant cells.

Fibrin and Fibrinolysis in Cancer.

In 1878, Billroth discovered that tumor cells invest themselves in a fibrin thrombus, and he hypothesized that fibrin promotes tumor growth and invasion. Since then, many observations have supported this concept, showing that many hemostatic factors including fibrinogen, fibrin, and components of the fibrinolytic system have indeed a complex interaction with cancer growth and metastasis. Fibrin promotes cell migration by providing a matrix for tumor cell migration and by interactions with adhesive molecules and integrins. Fibrin-containing vascular endothelial growth factor promotes angiogenesis. Fibrin interacts with platelets and leukocytes, and promotes their respective carcinogenic properties. Fibrinolytic components exert different effects on tumors. Plasmin activates latent growth factors, and breaks down extracellular matrix (ECM), while urokinase plasminogen activator (uPA) and the uPA receptor (uPAR) form complexes with vitronectin and integrins to promote tumor cells to adhere to the ECM. This complex also binds the epidermal growth factor receptor on the tumor cell membrane, and signals the RAF-MEK-ERK pathway. The complex also binds to the G protein-coupled receptors leading to cell proliferation. Plasminogen activator inhibitor 1 (PAI-1) inhibits apoptosis, and increases tumor cell survival. PAI-1 also enhances cell senescence, leading to production of tumorigenic cytokines by the senescence secretome. The presence of uPA/uPAR and PAI-1 represents a strong biomarker for tumor aggressiveness and poor prognosis. Multiple attempts by blocking various carcinogenic steps have shown tumor-suppressing effects in experimental animals, but human responses are uncertain without clinical trials.

Trabectedin modulates the senescence-associated secretory phenotype and promotes cell death in senescent tumor cells by targeting NF-κB.

Therapy-induced senescence is a major cellular response to chemotherapy in solid tumors. Senescent tumor cells acquire a secretory phenotype, or SASP, and produce pro-inflammatory factors, whose expression is largely under NF-κB transcriptional control. Secreted factors play a positive role in driving antitumor immunity, but also exert negative influences on the microenvironment, and promote tumor growth and metastasis. Moreover, subsets of cancer cells can escape the senescence arrest, driving tumor recurrence after treatments. Hence, removal the senescent tumor cells, or reprogramming of the senescent secretome, have become attractive therapeutic options.The marine drug trabectedin was shown to inhibit the production of pro-inflammatory mediators by tumor-infiltrating immune cells and by myxoid liposarcoma cells. Here, we demonstrate that trabectedin inhibits the SASP, thus limiting the pro-tumoral activities of senescent tumor cells in vitro. We show that trabectedin modulates NF-κB transcriptional activity in senescent tumor cells. This results in disruption of the balance between antiapoptotic and proapoptotic signals, and sensitization of cells to Fas-mediated apoptosis. Further, we found that trabectedin inhibits escape from therapy-induced senescence, at concentrations that do not affect the viability of bulk tumor population.Overall, our data demonstrate that trabectedin has the potential to inhibit multiple detrimental effects of therapy-induced senescence.