Senescence In Breast Cancer Cells Research Articles

Abstract 2552: Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses

Abstract Cancer immunotherapies have shown remarkable clinical activity across several tumor types but responses have been modest in breast cancers. This is attributed to low levels of neo-antigens in breast tumors and immunosuppressive nature of mammary tumor microenvironment. Here we investigated if we can counteract these obstacles to an effective immunotherapy by inducing senescence in breast cancer cells. Senescence is one of the physiological stress response programs characterized by loss of proliferative potential and secretion of a variety of pro-inflammatory markers which promotes identification and removal of senescent cells by the immune cells. Here we hypothesized that senescence will stimulate anti-tumor immune response by establishing immune-permissive microenvironment. We utilized immunocompetent and humanized PDX-bearing mice to investigate senescence-induced modulation of tumor secretome proteome, transcriptome and immune cell recruitment and activity. An FDA-approved small molecule CDK4/6 inhibitor was used to induce senescence. We detected induction of multiple markers of senescence after CDK4/6 inhibitor treatment of cells and tumors. Senescent cells and tumors had increased secretion of chemokines CCL4, CCL5, CXCL9, CXCL10 and CXCL11 which facilitated T cell homing into the tumor. This was a result of their transcriptional induction by NF-κB. Consequently, we observed the dynamic enrichment of tumor immune microenvironment with T cells in tumors treated with CDK4/6 inhibitor. Furthermore, the recruitment of adoptively transferred ex-vivo activated anti-tumor T cells was significantly enhanced in tumors that were treated with CDK4/6 inhibitor. Finally, CDK4/6 inhibitor-treated tumors became responsive to T cell-activating immunotherapy with antibody agonists of T cell co-stimulatory receptors OX40 and 4-1BB. In summary, our findings suggest that CDK4/6 inhibition can facilitate T cell recruitment into the mammary tumors which can lead to an improved immunotherapy response. This pre-clinical study provides rationale for clinical development of CDK4/6 inhibitors and T cell agonists and uncovers the potential of senescence-inducing approach for sensitizing tumors to immune therapy. Citation Format: Ashlyn Blevins, Stacey Mont, Hunter Lawrence, Nabil Saleh, Sheau-Chiann Chen, Ann Richmond, Anna Vilgelm. Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2552.

17-β estradiol promotes autophagy and induces cellular senescence in breast cancer cells.

e12523 Background: The fact that estrogen (17-β estradiol or E2) is a known carcinogen notwithstanding, mounting evidence suggest that E2 has the potential to exert anti-cancer effects against various forms of cancer. Using in vitro models we, and others, have previously demonstrated that E2 disrupts intracellular iron metabolism in such a way that arrests cell cycling in breast and ovarian cancer cells. However, the cellular and molecular correlates underlying this cytostatic effect of E2 in cancer cells remain elusive. Methods: In this study, metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer cells treated with 20 nM E2 were assessed for mitochondrial function, cell proliferation, apoptosis and senescence at different time points post treatment. Results: E2 treatment resulted in a significant mitochondrial membrane depolarization; an outcome that associated with a significant loss of mitochondrial function and the accumulation of auto-phagosomes. It also significantly upregulated the expression of the cell cycle regulating cyclin-dependent kinase inhibitor, p21 protein and enhanced the activation (de-phosphorylation) of the tumour suppressor retinoblastoma (Rb) protein. Although, as previously shown, E2 did not induced classis apoptosis; it resulted in a significant elevation in senescence-associated β- galactosidase levels. Conclusions: In summary, these findings suggest that E2 treatment mediates its anti-cancer potential by disrupting mitochondrial function and precipitating autophagy and cell senescence.

Dynamic Metabolic Response to Adriamycin-Induced Senescence in Breast Cancer Cells.

Cellular senescence displays a heterogeneous set of phenotypes linked to tumor suppression; however, after drug treatment, senescence may also be involved in stable or recurrent cancer. Metabolic changes during senescence can provide detailed information on cellular status and may also have implications for the development of effective treatment strategies. The metabolic response to Adriamycin (ADR) treatment, which causes senescence as well as cell death, was obtained with the aid of metabolic profiling and isotope tracing in two human breast cancer cell lines, MCF7 and MDA-MB-231. After 5 days of ADR treatment, more than 60% of remaining, intact cells entered into a senescent state, characterized by enlarged and flattened morphology and positive blue staining using SA-β-gal. Metabolic trajectory analysis showed that the two cell lines’ responses were significantly different and were divided into two distinct stages. The metabolic shift from the first stage to the second was reflected by a partial recovery of the TCA cycle, as well as amino acid and lipid metabolisms. Isotope tracing analysis indicated that the higher level of glutamine metabolism helped maintain senescence. The results suggest that the dynamic changes during senescence indicate a multi-step process involving important metabolic pathways which might allow breast cancer cells to adapt to persistent ADR treatment, while the higher level of anapleurosis may be important for maintaining the senescent state. Ultimately, a better understanding of metabolic changes during senescence might provide targets for cancer therapy and tumor eradication.

Abstract 318: Inhibition of FASN induces apoptosis and senescence in breast cancer

Abstract Fatty Acid Synthase (FASN), the key multienzyme in de novo lipogenesis, is upregulated and activated in numerous carcinomas, including breast cancer. Fatty acids play a fundamental role in cellular structure, energy production and storage and are intermediates in the biosynthesis of hormones and other biologic molecules. Therefore, FASN is a novel therapeutic target for treatment in cancer cells that have upregulated FASN protein. BT474 breast cancer cells, which overexpress FASN, were treated with a pharmacologic FASN inhibitor and resulted in apoptosis in a dose-dependent manner. Inhibition of FASN simultaneously upregulates the pro-apoptotic BCL2 family members Bim, Puma, and Noxa; additionally, FASN inhibition alters the NDAPH/NADP+ balance and induces reactive oxygen species (ROS) production. Most significantly, FASN inhibition induces sensitization of breast cancer cells to the BH3 mimetic navitoclax, a potential opportunity to use BH3 mimetics for the treatment of breast carcinomas. These results support further preclinical and potentially clinical development of a dual FASN and Bcl-2 treatment for breast cancer tumors that express FASN. In addition to apoptosis, there is an increase in cellular senescence in breast cancer cells overexpressing FASN when treated with FASN inhibitors. The increase in ROS, a known effect of FASN inhibition, is known to induce cellular stress, resulting in both apoptosis and senescence. Increased senescence was initially observed by x-gal staining of BT474 cells treated with FASN inhibitors via microscopy and FLOW. We also observed an increased expression of several pro-senescent genes both at the protein and mRNA levels, including p16/INK4a, p14ARF/p19ARF, p15, and p21. A hallmark of age-induced cellular senescence is the shortening of telomeres. However, telomere shortening was not observed, suggesting that senescence was a result of FASN inhibition and activation of senescence-induced genes. Therefore, inhibition of FASN appears to be an ideal target for therapeutic treatment of breast cancers that overexpress FASN through both apoptosis and senescence. Citation Format: Travis Vander Steen, Ruth Lupu. Inhibition of FASN induces apoptosis and senescence in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 318.

MIAT lncRNA is overexpressed in breast cancer and its inhibition triggers senescence and G1 arrest in MCF7 cell line.

Long non-coding RNAs are known as key regulators in the progression and metastasis of breast cancer. MIAT originally has been considered as an lncRNA to be associated with a susceptibility to myocardial infarction. Here, we have detected the expression of MIAT in different cancer cells and a series of breast tumor tissue. MIAT expression was much higher in high-grade tumors compared to low-grade ones. Unlike P53 positive tumors, MIAT expression was upregulated in ER, PR, Her2 positive tumor tissues. Knockdown MIAT suppressed breast cancer cell proliferation and caused G1 arrest in cell cycle. Furthermore, downregulation of MIAT promoted apoptosis and significantly decreased migration of breast cancer cells. An increase in the expression of mir-302, mir-150, and a decrease in the expression of mir-29c were detected following MIAT silencing. More importantly, knockdown MIAT significantly elevated the expression of p16Ink4A and Cox2, which commitment cellular senescence in breast cancer cells. Altogether, our results suggest that MIAT involved in breast cancer progression and could be candidate as a novel tumor marker for diagnosis and treatment of breast cancer.

Oct4 suppresses IR‑induced premature senescence in breast cancer cells through STAT3- and NF‑κB-mediated IL‑24 production.

Breast cancer stem cells (BCSCs) are a small subpopulation of breast cancer cells that have been proposed to be a primary cause of failure of therapies, including ionizing radiation (IR). Their embryonic stem-like signature is associated with poor clinical outcome. In the present study, the function of octamer-binding transcription factor 4 (Oct4), an embryonic stem cell factor, in the resistance of BCSCs to IR was investigated. Mammosphere cells exhibited increased expression of stemness-associated genes, including Oct4 and sex-determining region Y-box 2 (Sox2), and were more resistant to IR compared with serum-cultured monolayer cells. IR-resistant MCF7 cells also exhibited significantly increased expression of Oct4. To investigate the possible involvement of Oct4 in IR resistance of breast cancer cells, cells were transfected with Oct4. Ectopic expression of Oct4 increased the clonogenic survival of MCF7 cells following IR, which was reversed by treatment with small interfering RNA (siRNA) targeting Oct4. Oct4 expression decreased phosphorylated histone H2AX (γ-H2AX) focus formation and suppressed IR-induced premature senescence in these cells. Mammosphere, IR-resistant and Oct4-overexpressing MCF7 cells exhibited enhanced phosphorylation of signal transducer and activation of transcription 3 (STAT3) (Tyr705) and inhibitor of nuclear factor κB (NF-κB), and blockade of these pathways with siRNA against STAT3 and/or specific inhibitors of STAT3 and NF-κB significantly increased IR-induced senescence. Secretome analysis revealed that Oct4 upregulated interleukin 24 (IL-24) expression through STAT3 and NF-κB signaling, and siRNA against IL-24 increased IR-induced senescence, whereas recombinant human IL-24 suppressed it. The results of the present study indicated that Oct4 confers IR resistance on breast cancer cells by suppressing IR-induced premature senescence through STAT3- and NF-κB-mediated IL-24 production.

SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex

BackgroundSALL1 is a multi-zinc finger transcription factor that regulates organogenesis and stem cell development, but the role of SALL1 in tumor biology and tumorigenesis remains largely unknown.MethodsWe analyzed SALL1 expression levels in human and murine breast cancer cells as well as cancer tissues from different types of breast cancer patients. Using both in vitro co-culture system and in vivo breast tumor models, we investigated how SALL1 expression in breast cancer cells affects tumor cell growth and proliferation, metastasis, and cell fate. Using the gain-of function and loss-of-function strategies, we dissected the molecular mechanism responsible for SALL1 tumor suppressor functions.ResultsWe demonstrated that SALL1 functions as a tumor suppressor in breast cancer, which is significantly down-regulated in the basal like breast cancer and in estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) triple negative breast cancer patients. SALL1 expression in human and murine breast cancer cells inhibited cancer cell growth and proliferation, metastasis, and promoted cell cycle arrest. Knockdown of SALL1 in breast cancer cells promoted cancer cell growth, proliferation, and colony formation. Our studies revealed that tumor suppression was mediated by recruitment of the Nucleosome Remodeling and Deacetylase (NuRD) complex by SALL1, which promoted cancer cell senescence. We further demonstrated that the mechanism of inhibition of breast cancer cell growth and invasion by SALL1-NuRD depends on the p38 MAPK, ERK1/2, and mTOR signaling pathways.ConclusionOur studies indicate that the developmental control gene SALL1 plays a critical role in tumor suppression by recruiting the NuRD complex and thereby inducing cell senescence in breast cancer cells.

TGF-beta receptor mediated telomerase inhibition, telomere shortening and breast cancer cell senescence

ABSTRACTHuman telomerase reverse transcriptase (hTERT) plays a central role in telomere lengthening for continuous cell proliferation, but it remains unclear how extracellular cues regulate telomerase lengthening of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces the hTERT gene repression in a BMPRII receptor- and Smad3-dependent manner in human breast cancer cells. Chonic exposure of human breast cancer cells to BMP7 results in short telomeres, cell senescence and apoptosis. Mutation of the BMPRII receptor, but not TGFbRII, ACTRIIA or ACTRIIB receptor, inhibits BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres and continued cell proliferation. Expression of hTERT prevents BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging by a mechanism involving BMPRII receptor- and Smad3-mediated repression of the hTERT gene.

Abstract 5105: Exosomes secreted by proinflammatory macrophages modulate breast cancer

Abstract Multiple cell types reside within the tumor microenvironment (TME) including cancer stem cells, stromal cells, and inflammatory cells. Cellular crosstalk is initiated between these cells through a variety of secreted biomolecules such as cytokines, growth factors, and exosomes. The role of inflammation and numerous secretory factors within the TME have been implicated in the development and progression of various cancers including breast. In this study, we wanted to evaluate the effect of macrophages (a major constituent of inflammation) on breast cancer development. Our study focused on macrophage secreted exosomes which are membraneous nanovesicles that contain various miRNA, mRNA, and proteins. We hypothesized that macrophage secreted exosomes will modulate breast cancer phenotype. An in vitro cell culture system was used in this study to examine macrophage exosome treatments on breast cancer cell lines MCF-7 (ER+) and MDA-MB 231 (ER-, PR-, HER2/neu-). Human monocytic THP-1 monocytes were activated with a common phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) and conditioned media (CM) was collected following 48 h. Exosomes were isolated from THP-1 CM via the use of an exosome isolation reagent and standard centrifugation. Breast cancer cells were then treated with THP-1 exosomes and cell morphology, proliferation, MEK/ERK and PI3K/AKT signaling pathways, and cell cycle were evaluated. MCF-7 and MDA-MB 231 exhibited round and spindle-like morphologies as well as a significant decrease in proliferation at 24 h treatments. Furthermore, MDA-MB 231 displayed increased p-ERK, while MCF-7 showed increased p-MEK following Western blot analysis. Both cell lines exhibited decreased p-AKT and p-S6 kinase expression. Cell cycle analysis showed that MCF-7 and MDA-MB 231 arrested at G2/M at 24 h exosome treatments. THP-1 exosome treatments led to a significant increase in MDA-MB 231 senescence as determined by a senescence assay looking at senescence-associated beta-galactosidase. Increased p21 and decreased Cdc2/Cyclin B1 was also observed which indicates a G2/M arrest. We conclude that specific macrophage secreted exosomes may induce breast cancer cell senescence and affect breast cancer progression. Citation Format: Robert Bronislaw Bednarczyk, Neha Y. Tuli, Ghada Ben Rahoma, Rachana Maniyar, Abraham Mittelman, Raj K. Tiwari. Exosomes secreted by proinflammatory macrophages modulate breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5105.

Abstract P2-06-10: Loss of COX5B inhibits proliferation and promotes senescence via mitochondrial dysfunction in breast cancer

Abstract Background: COX5B, a peripheral subunit of the cytochrome c oxidase complex, is reported to maintain the stability of this complex and impact on cell viability. Recently, several studies demonstrated that cox5b may involve in cancer progression. In our study, we also found that COX5B was upregulated both in breast cancer tissues and cells. However, the role and mechanism of COX5B in breast cancer remain unclear. Therefore, the aim of this study is to validate COX5B functions and its underlying mechanisms in breast cancer cells. Methods: SILAC (stable isotope labeling with amino acids in cell culture) assay was conducted in mammary epithelial cells and its paired breast tumor cells, and the results was validated by other two paired tissues. Forty tumors and twenty normal or benign samples were implied for COX5B expression by IHC. Online Kaplan Meier plotting tool was used to confirm the association between COX5B expression and clinical outcome. Three Cell lines (MDA-MB-231, MDA-MB-468 and MCF-7) were constructed by sh-RNA and the efficiency was confirmed by qPCR and western blot and then migration, proliferation and senescence characteristics were evaluated. Furthermore, microarray was implemented to detect potential mechanisms and the associated genes were assessed by qPCR. ELISA was carried out for IL8 secretion. Condition medium was collected from stable knock down cells and used as attractant in lower chamber of tranwell assay to examine microenvironment influence. Finally, the intracellular ATP glucose uptake and lactate secretion were evaluated, as well as mitochondrial membrane potential and ROS production. Results: We found that the level of COX5B was elevated in breast cancer and cell lines. Patients with high levels of COX5B were likely to have a longer disease-free survival than patients with low levels. Furthermore, loss of COX5B inhibited proliferation and induced the senescence of breast cancer cells, which was accompanied by increases in the secretion of IL-8 and other cytokines. Additionally, conditioned medium form COX5B knockdown cells increased the migration of breast cancer cells compared with conditioned medium from control cells. The mechanisms underlying these processes were associated with mitochondrial dysfunction due to increased ROS production and decreased MMP and metabolism disorder. Conclusion: Our results show that loss of COX5B inhibits proliferation and promotes senescence in breast cancer cells, which is associated with mitochondrial dysfunction. Indeed, loss of COX5B has different functions in target cells and the surrounding environments. These findings may provide a new perspective for the design of anti-cancer therapy combining with anti-IL8 treatment. Citation Format: Gao S-P, Sun H-F, Jiang H-L, Li L-D, Jin W. Loss of COX5B inhibits proliferation and promotes senescence via mitochondrial dysfunction in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-10.

The dual function of PRMT1 in modulating epithelial-mesenchymal transition and cellular senescence in breast cancer cells through regulation of ZEB1.

Although the involvement of protein arginine methyltransferase 1 (PRMT1) in tumorigenesis has been reported, its roles in breast cancer progression and metastasis has not been elucidated. Here we identified PRMT1 as a key regulator of the epithelial-mesenchymal transition (EMT) in breast cancer. We showed that the EMT program induced by PRMT1 endowed the human mammary epithelial cells with cancer stem cell properties. Moreover, PRMT1 promoted the migratory and invasive behaviors in breast cancer cells. We also demonstrated that abrogation of PRMT1 expression in breast cancer cells abated metastasis in vivo in mouse model. In addition, knockdown of PRMT1 arrested cell growth in G1 tetraploidy and induced cellular senescence. Mechanistically, PRMT1 impacted EMT process and cellular senescence by mediating the asymmetric dimethylation of arginine 3 of histone H4 (H4R3me2as) at the ZEB1 promoter to activate its transcription, indicating the essential roles of this epigenetic control both in EMT and in senescence. Thus, we unraveled a dual function of PRMT1 in modulation of both EMT and senescence via regulating ZEB1. This finding points to the potent value of PRMT1 as a dual therapeutic target for preventing metastasis and for inhibiting cancer cell growth in malignant breast cancer patients.

Abstract 1426: Lung epithelial cells induce both phenotype alteration and senescence in breast cancer cells

Abstract Purpose: Breast cancer is the most common cancer in women. The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells. Methods: Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype. Results: Co-culture of the breast cancer cells had different effects on breast cancer cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slight more mesenchymal spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin express in MDA-MB-231 using shRNA prevented this phenotypic reversion in coculture. In addition, we had noted enlarged MDA-MB-231 cells in the co-cultures with SAEC and NHBE but not BEAS-2B cells as suggestion of senescence. A subset of these cells stained with the senescence associated marker β-galactosidase (SA-β-gal). Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture. Conclusions: Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells by changing their phenotypes.Similar to the hepatocytes, these normal cells may promote the survival, seeding and even dormancy of aggressive mesenchymal carcinoma cells, with phenotypes similar to MDA-MB-231 cells, but also provide progressive signaling when stressed accounting for the spindle morphology and slightly increased growth rates of the epithelial carcinoma cells, such as those MCF-7 represents. In this latter manner, insults the small airway may provoke an outgrowth from dormant breast cancer cells. Citation Format: Masashi Furukawa, Alan Wells, Sarah Wheeler, Amanda M. Clark. Lung epithelial cells induce both phenotype alteration and senescence in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1426. doi:10.1158/1538-7445.AM2015-1426

Abstract 3101: In-vitro characterization of Abemaciclib pharmacology in ER+ breast cancer cell lines

Abstract Dysregulation of the cell-cycle is a hallmark of cancer and genetic alterations in its regulatory machinery (or checkpoints) occur in most human tumors. The majority these defects are found in genes encoding for proteins regulating G1 phase progression, such as Rb, E2F1, CyclinD1, CDK4 and CDK6. Aberrant regulation of the G1 kinases CDK4 and CDK6, as well as overexpression or gene amplification of CyclinD, lead to inhibition of tumor suppressors such as Rb resulting in an accelerated cell cycle progression. Alterations in the CyclinD-CDK4/6-Rb pathway are common in breast cancer. Amplification of CCND1 gene encoding CyclinD1, occurs in 15% to 20% of breast cancers, and CyclinD1 overexpression is even more common (up to 50% of breast cancers). Abemaciclib is a reversible, ATP competitive, kinase inhibitor selective for CDK4 and CDK6 that has been shown to prevent growth of malignant cells in-vitro and in-vivo. This antitumor activity is mediated by inhibiting the phosphorylation of Rb and subsequent blockade of tumor cell cycle progression through G1/S. CDK4/6 inhibitors in general have shown significant potential for the treatment of metastatic breast cancer and Abemaciclib, in particular, is currently being evaluated in advanced clinical trials (Phase II as single agent and Phase III in combination with anti-hormone therapy) in hormone receptor positive metastatic breast cancer patients. The goal of this study was to investigate the mechanism of action of Abemaciclib in ER+ luminal breast cancer. We have evaluated the response of the drug in a diversity of breast cancer cell lines. Phenotypic characterization of sensitive cell lines was carried out by monitoring proliferation, cell cycle progression and phosphorylation of Rb using High Content Imaging. Senescence markers were included in the study to monitor the final outcome of the cells upon sustained exposure to the drug. Luminal ER+ breast cancer cells showed a marked sensitivity to treatment with Abemaciclib with IC50 values ranging from 5nM to 2uM. Simultaneous decrease in Rb phosphorylation with sustained accumulation of the 2N subpopulation was observed. Associated to the G1S arrest phenotype, Abemaciclib treatment resulted in a decrease of cell proliferation markers (Ki67 and BrdU). Additionally, a marked hyper-methylation profile (Histone H3K9met3) and a decrease of FOXM1 expression were observed, as well as an accumulation of endogenous beta-galactosidase and p21. Taken together this profile suggests that Abemaciclib acts through promotion of senescence in breast cancer cells. Abemaciclib prevents proliferation of breast cancer cell lines expressing D-types cyclins by promoting cell cycle arrest mediated by inhibition of Rb phosphorylation. Abemaciclib is a CDK4/6 inhibitor with potential to treat breast cancer by blocking cell proliferation leading to induction of senescence. Citation Format: Maria Jose Lallena, Karsten Boehnke, Raquel Torres, Ana Hermoso, Joaquin Amat, Bruna Calsina, Alfonso De Dios, Sean Buchanan, Jian Du, Richard Paul Beckmann, Xueqian Gong, Ann Mcnulty. In-vitro characterization of Abemaciclib pharmacology in ER+ breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3101. doi:10.1158/1538-7445.AM2015-3101

Abstract 1333: CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor apoptosis in breast cancer

Abstract Background: The therapeutic benefit of blocking HER2/HER3 signaling in breast cancer (BC) has been demonstrated by several studies. We have previously shown that the pleiotropic T-helper type 1 (Th1) cytokines IFN-γ and TNF-α induce senescence in BC cells and that all BC cell lines tested express IFN-γ and TNF-α receptors by western blot analysis. We have also demonstrated an inverse correlation between the HER2 expression level and the senescence induced by the treatment with both cytokines. Moreover, simultaneous HER-2/HER-3 blockade significantly enhanced cytokine-induced senescence. Here, we studied whether these Th1 cytokines induce apoptosis of HER-2 expressing BC cells and assessed the impact of IFN-γ and TNF-α with simultaneous HER-2 and HER-3 blockade on permanent tumor abrogation. Results: To determine the Th1-mediated effects on HER2high (SK-BR-3, BT-474), HER2intermediate (MCF-7, T-47D), and HER2low (MDA-MB-231) human BC cell lines in vitro, we performed co-culture of increasing number of HER2 Class II peptide-specific CD4+ T-cells (generated by priming CD4+ T cells with HER2 peptide loaded type-1 polarized DCs) with BC cells using a transwell culture system. This resulted in a cell number-dependent apoptosis of SK-BR-3 and MCF-7, but not MDA-MB-231 cells compared with CD4+ T cells primed either with immature dendritic cells (DC) or mature DC plus irrelevant (Class II BRAF) peptide. In addition, SK-BR-3 cells incubated with supernatants from the CD4+ T cells-immature DC or mature DC co-culture demonstrated similar results. Compared with controls, HER2-specific Th1 cells generated a 25-fold increase in SK-BR-3 apoptosis by DAPI staining. Neutralizing antibodies against IFN-γ and TNF-α significantly reduced apoptosis induction. Also, IFN-γ and TNF-α treatment resulted in significant apoptosis of SK-BR-3 and MCF-7, but not MDA-MB-231 cells. However, MDA-MB-231 cells transfected with a wild type HER2 plasmid, were highly susceptible to cytokine-induced apoptosis. Treatment of HER-2-depleted cells (by RNAi) with IFN-γ and TNF-α resulted in an increased apoptotic phenotype. Although the combined treatment of IFN-γ and TNF-α in HER3-depleted cells did not enhanced the apoptosis, the double knock down with HER2 and HER3 RNAi strongly increased apoptosis induction as observed by western blot analysis of active caspase-3 and flow cytometric analysis of Anexin V-PI staining. Interestingly, the double depleted cells treated with IFN-γ alone evidenced slightly higher cleaved caspase-3 levels than TNF-α alone but the combined treatment with both cytokines had a strong synergistic effect. Conclusions: Our results establish a role for IFN-γ and TNF-α in inducing tumor apoptosis in BC. An effective CD4 Th1 response, combined with HER-2 and HER-3 blockade can significantly drive tumor apoptosis that can be explored as treatment to effectively eliminate residual BC cells and prevent recurrence. Citation Format: Cinthia Rosemblit, Jashodeep Datta, Erik Berk, Brian J. Czerniecki. CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor apoptosis in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1333. doi:10.1158/1538-7445.AM2015-1333

Retinoids induce cellular senescence in breast cancer cells by RAR-β dependent and independent pathways: Potential clinical implications (Review).

Most studies on cellular senescence (CS) have been performed in vitro by employing cytotoxic agents, irradiation, chromatin and telomerase modulators or by activating certain oncogenes. All these approaches usually lead to DNA damage, gene instability and/or chromatin alterations that primarily affect p53-p21 signaling. Little is known on whether retinoids and rexinoids, which are cell differentiation agents, can also induce CS in vitro and in vivo, and which molecular mechanisms are involved in promoting the senescent phenotype. We reviewed the recent publications on CS induced by retinoids and rexinoids in ER+ and ER− breast cancer cell lines and in corresponding animal models of mammary carcinogenesis which simulate those of human breast cancer. The role of retinoic acid receptors β2 and 5 (RARβ2 and RARβ5) and of receptor independent genes involved in mediating the senescence program of retinoids and rexinoids in ER+ and ER− breast cancer cells is discussed. Potential strategists for clinical implication of CS as biomarker of prognosis and of response to treatment with retinoids, rexinoids and with other cell differentiation and antitumor agents are outlined.

Abstract P6-05-03: CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor senescence in breast cancer

Abstract Background: HER-2 a molecular oncodriver in breast tumorigenesis is over expressed in 25% of human breast cancers, and its expression correlates with enhanced tumor aggressiveness. While targeted therapies have improved outcomes many patients become resistant or recur. We have recently established a progressive loss in anti-HER-2 CD4 Th1 responses during disease progression and is associated with outcomes. The pleiotropic Th1 cytokines IFN-γ and TNF-α have diverse effects on tumor epithelial cells. In this study we sought to determine whether these Th1 cytokines induce senescence of HER-2 expressing breast cancer cells and assess the impact of IFN-γ and TNF-α with simultaneous HER-2 and HER-3 blockade. Results: All breast cancer cell lines tested express IFN-γ and TNF-α receptors measured by western blot analysis. The high and intermediate HER-2 expressing cells are sensitive to tumor senescence induction when treated with combinations of IFN-γ and TNF-α in a dose dependent manner. Low HER-2 expressing cells were less sensitive to senescence induction as measured by positive β-galactosidase activity and the expression of p15INK4b and p16INK4a by western blot. Addition of IFN-γ and TNF-α treatment to HER-2-depleted cells by RNAi resulted in an increase senescent phenotype and was increased further when the cells were double HER-2- and HER-3-depleted. To determine whether CD4 Th1 mediated effects on high HER-2 human breast cancer cell lines, we co-cultured increasing number of HER-2 antigen-primed CD4+ T cells with high HER-2 human breast cancer cells in a transwell cell culture system. This resulted in a dose-dependent senescence of breast cancer cells compared with CD4+ T cells primed either with immature dendritic cells (DC) or mature DC plus irrelevant (Class II BRAF) peptide. In addition, SK-BR-3 breast cancer cells incubated with the supernatant of the CD4+ T cells-immature DC or mature DC co-culture demonstrated similar results. CD4+ Th1-elaborated cytokines IFN-γ and TNF-α in the supernatants were confirmed using ELISA. Blocking antibodies against IFN-γ and TNF-α demonstrated reduced senescence induction. Conclusions: Our results establish a role for IFN-γ and TNF-α in inducing tumor senescence in breast cancer. An effective CD4 Th1 responses combined with HER-2 and HER-3 blockade can significantly drive tumor senescence in breast cancer that can be explored as treatment to effectively eliminate residual breast cancer cells and prevent recurrence. Citation Format: Cinthia Rosemblit, Brian J Czerniecki. CD4 Th1 cytokines and HER-2/HER-3 blockade induces tumor senescence in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-05-03.

Lung epithelial cells induce both phenotype alteration and senescence in breast cancer cells.

PurposeThe lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells.MethodsCo-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype.ResultsCo-culture of the breast cancer cells with the normal lung cells had different effects on the epithelial and mesenchymal carcinoma cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slightly more mesenchymal-spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin expressed in MDA-MB-231 using shRNA prevented this phenotypic reversion in co-culture. Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture.ConclusionsOur data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells coincident with changing their phenotypes.

PLK1 Inhibition Down-regulates Polycomb Group Protein BMI1 via Modulation of the miR-200c/141 Cluster

The polycomb group protein BMI1 is an important regulator of cancer stem cell (CSC) phenotype and is often overexpressed in cancer cells. Its overexpression leads to increase in CSC fraction and therapy resistance in tumors. BMI1 functions via polycomb repressive complex 1 (PRC1)-mediated gene silencing and also via PRC1-independent transcriptional activities. At present, very little is known about the therapy reagents that can efficiently inhibit BMI1 expression, and the CSC phenotype. Here, we report that the polo-like kinase 1 (PLK1) regulates BMI1 expression, and that its inhibition can efficiently down-regulate BMI1 expression and PRC1 activity, and induce premature senescence in breast cancer cells. We also show that the exogenous BMI1 overexpression mitigates anti-oncogenic effects of PLK1 inhibition and overcomes senescence induction by PLK1 inhibitors. We further show that PLK1 inhibition down-regulates BMI1 by upregulating the miRNA-200c/141 cluster, which encodes miR-200c and miR-141, both of which are known to post-transcriptionally downregulate BMI1 expression. Thus, our data suggest that PLK1 inhibitors can be successfully used to inhibit growth of tumors in which PcG protein BMI1 is overexpressed or the PRC1 activity is deregulated.

Abstract 2249: N- and C-terminal peptides of the tumor suppressor protein IGFBP7 differentially induce growth arrest or senescence in breast cancer cells

Abstract N- and C-terminal peptides of the tumor suppressor protein IGFBP7 differentially induce growth arrest or senescence in breast cancer cells Tania Benatar, Yutaka Amemiya, Valentina Evdokimova, Wenyi Yang and Arun Seth We have previously shown that insulin-like growth factor binding protein 7 (IGFBP7) is a tumor suppressor in breast cancer. IGFBP7 treatment resulted in breast cancer cell growth inhibition via induction of senescence and apoptotic pathways. Xenografted tumors overexpressing IGFBP7 were significantly growth-impaired as were the xenografted tumors treated systemically with purified IGFBP7. Given its low toxicity and high selectivity towards cancerous tissues, IGFBP7 is considered to be a promising anticancer agent. Its anticancer efficacy was further strengthened by our recent findings showing that the N-terminal 97 amino acid IGFBP7 domain is required for blocking Insulin-like Growth Factor 1 Receptor (IGF1R) activity and downstream PI3K-AKT-mTOR signaling. To further identify parameters that could predict IGFBP7-responsiveness in breast tumors, a variety of breast cancer cell lines were examined for growth responses and signaling pathways in response to IGFBP7 treatment. In this study, we found that treatment of some aggressive breast cancer cell lines, with IGFBP7 resulted in its proteolytic cleavage between Lys-97 and Ala-98 thus creating N-terminal and C-terminal specific peptides. While both the N- and C-terminal peptides were capable of growth inhibition, only the C-terminal fragment was efficient at inducing cellular senescence. The IGFBP7-responsiveness of breast cancer cell lines was a direct consequence of their ability to process IGFBP7. In order to compare which genes and pathways arewere affected by IGFBP7-full length (FL) versus cleaved form (CF) overexpression, we performed transcriptome Next- Generation sequencing from parental MDA-MB-468, MDA-MB-468 overexpressing full length (FL) of IGFBP7-FL or overexpressing cleaved form (CF) of IGFBP7-CF. We obtained 2486 gene differences for FL expressers and 3953 differences for CF expressers using mapped reads with a quality of 10 and at least a two-fold change. Of the thirteen breast cancer-associated genes that were differentially expressed between MDA-MB-468 cells and FL or CF expressing cells, most (10/13) were further enhanced upon IGFBP7-CF overexpression. Taken together, Tthese results indicate that IGFBP7s distinct N- and C-terminal functional domains, are ideal candidates for the development of companion theranostiitcs, as the ability of the tumor to effectively cleave IGFBP7 will determine efficacy of treatment. Citation Format: Tania C. Benatar, Yutaka Amemiya, Valentina Evdokimova, Wenyi Yang, Arun Seth. N- and C-terminal peptides of the tumor suppressor protein IGFBP7 differentially induce growth arrest or senescence in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2249. doi:10.1158/1538-7445.AM2014-2249

Senescence associated inflammasome (SAI) activation promotes cancer progression (TUM4P.909)

Abstract Oncogene-induced senescence functions as a barrier against cell transformation and tumorigenesis. Paradoxically, recent studies indicate that senescence also promotes tumor progression through secretion of inflammatory molecules or senescence-associated secretory phenotype (SASP). However, the signaling pathways leading to SASP and the interaction between senescent cells and innate immune cells are poorly understood. Utilizing human and murine breast cancer cells, as well as breast cancer mouse models, this study demonstrates that senescence-associated inflammasome (SAI) generate a positive feedback loop to propagate inflammation through interaction with innate immune cells, contributing to the tumor development. Our data show that cellular senescence induced by oncogenes, such as K-Ras12V and Her2/ERBB2, led to inflammasome activation and IL-1beta production in both murine and human breast cancer cells. Notably, we found that blocking inflammasome activity could suppress both IL-1beta production and oncogene-induced senescence in breast cancer cells. Our results further show that the crosstalk between senescent cells and innate immune cells promoted inflammation and breast cancer progression. Together, our findings demonstrate a critical role for the senescence-associated inflammasome in oncogenic inflammation and tumor development.