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Supplementary Figures S1 - S10, Tables S1 - S2

Supplementary Data supporting the manuscript.

Cellular senescence entails an irreversible growth arrest that evolved in part to prevent cancer. Paradoxically, senescent cells secrete proinflammatory and growth-stimulatory molecules, termed the senescence-associated secretory phenotype (SASP), which is correlated with cancer cell proliferation in culture and xenograft models. However, at what tumor stage and how senescence and the SASP act on endogenous tumor growth in vivo is unknown. To understand the role of senescence in cancer etiology, we subjected p16-3MR transgenic mice, which permit the identification and selective elimination of senescent cells in vivo, to the well-established two-step protocol of squamous cell skin carcinoma, in which tumorigenesis is initiated by a carcinogen 7,12-dimethylbenz[α]anthracene, and then promoted by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that TPA promotes skin carcinogenesis by inducing senescence and a SASP. Systemic induction of senescence in nontumor-bearing p16-3MR mice using a chemotherapy followed by the two-step carcinogenesis protocol potentiated the conversion of benign papillomas to carcinomas by elevating p38MAPK and MAPK/ERK signaling. Ablation of senescent cells reduced p38MAPK and MAPK/ERK signaling, thereby preventing the progression of benign papillomas to carcinomas. Thus, we show for the first time that senescent cells are tumor promoters, not tumor initiators, and that they stimulate skin carcinogenesis by elevating p38MAPK and MAPK/ERK signaling. These findings pave the way for developing novel therapeutics against senescence-fueled cancers.Significance:These findings identify chemotherapy-induced senescence as a culprit behind tumor promotion, suggesting that elimination of senescent cells after chemotherapy may reduce occurrence of second cancers decades later.

AbstactAgeing is the largest risk factor for many chronic diseases. Studies of heterochronic parabiosis, substantiated by blood exchange and old plasma dilution, show that old-age-related factors are systemically propagated and have pro-geronic effects in young mice. However, the underlying mechanisms how bloodborne factors promote ageing remain largely unknown. Here, using heterochronic blood exchange in male mice, we show that aged mouse blood induces cell and tissue senescence in young animals after one single exchange. This induction of senescence is abrogated if old animals are treated with senolytic drugs before blood exchange, therefore attenuating the pro-geronic influence of old blood on young mice. Hence, cellular senescence is neither simply a response to stress and damage that increases with age, nor a chronological cell-intrinsic phenomenon. Instead, senescence quickly and robustly spreads to young mice from old blood. Clearing senescence cells that accumulate with age rejuvenates old circulating blood and improves the health of multiple tissues.

Wound healing is impaired with advanced age and certain chronic conditions, such as diabetes and obesity. Moreover, common cancer treatments, including chemotherapy and radiation, can cause unintended tissue damage and impair wound healing. Available wound care treatments are not always effective, as some wounds fail to heal or recur after treatment. Hence, a more thorough understanding of the pathophysiology of chronic, nonhealing wounds may offer new ideas for the development of effective wound care treatments. Cancers are sometimes referred to as wounds that never heal, sharing mechanisms similar to wound healing. We describe in this review how cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to chronic wounds versus cancer.

Cellular senescence entails an irreversible growth arrest that evolved in part to prevent cancer. Paradoxically, senescent cells secrete proinflammatory and growth-stimulatory molecules, termed the senescence-associated secretory phenotype (SASP), which is correlated with cancer cell proliferation in culture and xenograft models. However, at what tumor stage and how senescence and the SASP act on endogenous tumor growth in vivo is unknown. To understand the role of senescence in cancer etiology, we subjected p16-3MR transgenic mice, which permit the identification and selective elimination of senescent cells in vivo, to the well-established two-step protocol of squamous cell skin carcinoma, in which tumorigenesis is initiated by a carcinogen 7,12-dimethylbenz[α]anthracene, and then promoted by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We show that TPA promotes skin carcinogenesis by inducing senescence and a SASP. Systemic induction of senescence in nontumor-bearing p16-3MR mice using a chemotherapy followed by the two-step carcinogenesis protocol potentiated the conversion of benign papillomas to carcinomas by elevating p38MAPK and MAPK/ERK signaling. Ablation of senescent cells reduced p38MAPK and MAPK/ERK signaling, thereby preventing the progression of benign papillomas to carcinomas. Thus, we show for the first time that senescent cells are tumor promoters, not tumor initiators, and that they stimulate skin carcinogenesis by elevating p38MAPK and MAPK/ERK signaling. These findings pave the way for developing novel therapeutics against senescence-fueled cancers. Significance: These findings identify chemotherapy-induced senescence as a culprit behind tumor promotion, suggesting that elimination of senescent cells after chemotherapy may reduce occurrence of second cancers decades later.

Chronic exposure to UVR is known to disrupt tissue homeostasis, accelerate the onset of age-related phenotypes, and increase the risk for skin cancer-a phenomenon defined as photoaging. In this paper, we review the current knowledge on how UV exposure causes cells to prematurely enter cellular senescence. We describe the mechanisms contributing to the accumulation of senescent cells in the skin and how the persistence of cellular senescence can promote impaired regenerative capacity, chronic inflammation, and tumorigenesis associated with photoaging. We conclude by highlighting the potential of senolytic drugs in delaying the onset and progression of age-associated phenotypes in the skin.

Summary Cellular senescence is a stress or damage response that causes a permanent proliferative arrest and secretion of numerous factors with potent biological activities. This senescence-associated secretory phenotype (SASP) has been characterized largely for secreted proteins that participate in embryogenesis, wound healing, inflammation, and many age-related pathologies. By contrast, lipid components of the SASP are understudied. We show that senescent cells activate the biosynthesis of several oxylipins that promote segments of the SASP and reinforce the proliferative arrest. Notably, senescent cells synthesize and accumulate an unstudied intracellular prostaglandin, 1a,1b-dihomo-15-deoxy-delta-12,14-prostaglandin J2. Released 15-deoxy-delta-12,14-prostaglandin J2 is a biomarker of senolysis in culture and in vivo. This and other prostaglandin D2-related lipids promote the senescence arrest and SASP by activating RAS signaling. These data identify an important aspect of cellular senescence and a method to detect senolysis.

Processes that have been linked to aging and cancer include an inflammatory milieu driven by senescent cells. Senescent cells lose the ability to divide, essentially irreversibly, and secrete numerous proteases, cytokines and growth factors, termed the senescence-associated secretory phenotype (SASP). Senescent cells that lack p53 tumor suppressor function show an exaggerated SASP, suggesting the SASP is negatively controlled by p53. Here, we show that increased p53 activity caused by small molecule inhibitors of MDM2, which promotes p53 degradation, reduces inflammatory cytokine production by senescent cells. Upon treatment with the MDM2 inhibitors nutlin-3a or MI-63, human cells acquired a senescence-like growth arrest, but the arrest was reversible. Importantly, the inhibitors reduced expression of the signature SASP factors IL-6 and IL-1α by cells made senescent by genotoxic stimuli, and suppressed the ability of senescent fibroblasts to stimulate breast cancer cell aggressiveness. Our findings suggest that MDM2 inhibitors could reduce cancer progression in part by reducing the pro-inflammatory environment created by senescent cells.

Neurodegeneration is a major age-related pathology. Cognitive decline is characteristic of patients with Alzheimer's and related dementias and cancer patients after chemo- or radio-therapies. A recently emerged driver of these and other age-related pathologies is cellular senescence, a cell fate that entails a permanent cell cycle arrest and pro-inflammatory senescence-associated secretory phenotype (SASP). Although there is a link between inflammation and neurodegenerative diseases, there are many open questions regarding how cellular senescence affects neurodegenerative pathologies. Among the various cell types in the brain, astrocytes are the most abundant. Astrocytes have proliferative capacity and are essential for neuron survival. Here, we investigated the phenotype of primary human astrocytes made senescent by X-irradiation, and identified genes encoding glutamate and potassium transporters as specifically downregulated upon senescence. This down regulation led to neuronal cell death in co-culture assays. Unbiased RNA sequencing of transcripts expressed by non-senescent and senescent astrocytes confirmed that glutamate homeostasis pathway declines upon senescence. Our results suggest a key role for cellular senescence, particularly in astrocytes, in excitotoxicity, which may lead to neurodegeneration including Alzheimer's disease and related dementias.

SummaryCellular senescence is a stress or damage response that results in a permanent proliferative arrest and secretion of numerous factors with potent biological activities. This senescence-associated secretory phenotype (SASP) has been characterized largely for secreted proteins that participate in embryogenesis, wound healing, inflammation and many age-related pathologies. By contrast, lipid components of the SASP have been understudied. We show that senescent cells activate the biosynthesis of several eicosanoids that promote segments of the SASP and reinforce the proliferative arrest. Notably, senescent cells synthesize an unstudied intracellular prostaglandin, 1a,1b-dihomo-15-deoxy-delta-12,14-prostaglandin J2, which, together with other prostaglandin D2 derivatives, promote the senescence arrest and SASP by activating RAS signaling. Further, 15-deoxy-delta-12,14-prostaglandin J2 is a biomarker of senolysis in culture and in vivo. These data identify an important new aspect of cellular senescence and a novel method to detect senolysis.

Accumulation of senescent cells is associated with the progression of pulmonary fibrosis, but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM), lipid extracts, and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT, regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by antifibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR-90) induced profibrotic signaling in naive fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl LT in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cell removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5(+) cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16(Ink4a+)). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time to our knowledge that senescent cells secrete functional LTs, significantly contributing to the LT pool known to cause or exacerbate IPF.

Cellular senescence suppresses cancer by irreversibly arresting cell proliferation. Senescent cells acquire a proinflammatory senescence-associated secretory phenotype. Many genotoxic chemotherapies target proliferating cells nonspecifically, often with adverse reactions. In accord with prior work, we show that several chemotherapeutic drugs induce senescence of primary murine and human cells. Using a transgenic mouse that permits tracking and eliminating senescent cells, we show that therapy-induced senescent (TIS) cells persist and contribute to local and systemic inflammation. Eliminating TIS cells reduced several short- and long-term effects of the drugs, including bone marrow suppression, cardiac dysfunction, cancer recurrence, and physical activity and strength. Consistent with our findings in mice, the risk of chemotherapy-induced fatigue was significantly greater in humans with increased expression of a senescence marker in T cells prior to chemotherapy. These findings suggest that senescent cells can cause certain chemotherapy side effects, providing a new target to reduce the toxicity of anticancer treatments. Significance: Many genotoxic chemotherapies have debilitating side effects and also induce cellular senescence in normal tissues. The senescent cells remain chronically present where they can promote local and systemic inflammation that causes or exacerbates many side effects of the chemotherapy. Cancer Discov; 7(2); 165–76. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 115

The vitamin D receptor (VDR), and its ligand 1α,25-dihydroxyvitamin D3 (1,25D3) prevent breast cancer development and progression, yet the molecular mechanisms governing this are unclear. MicroRNAs (miRNAs) on the other hand, promote or inhibit breast cancer growth. To understand how VDR regulates miRNAs, we compared miRNA expression of wild-type (WT) and VDR knockout (VDRKO) breast cancer cells by a Mouse Breast Cancer miRNA PCR array. Compared to VDR WT cells, expressions of miR-214, miR-199a-3p and miR-199a-5p of the miR-199a/miR-214 cluster were 42, 15, and 10 fold higher in VDRKO cells respectively. Overexpression of VDR in breast cancer cells reduced the miR-199a/miR-214 cluster expression by 30%. VDR status also negatively correlated with Dnm3os expression, a non-coding RNA transcript of the dynamin-3 gene encoding the miR-199a/miR-214 cluster, suggesting that VDR represses this cluster through Dnm3os. Conversely, overexpression of miR-214 in MCF-7 and T47D cells antagonized VDR mediated signaling. Furthermore, there was a positive correlation between VDR status and the expression of Suppressor of fused gene (SuFu), a hedgehog pathway inhibitor. miR-214 on the other hand suppressed SuFu protein expression. These findings suggest a crosstalk between VDR and miR-214 in regulating hedgehog signaling in breast cancer cells, providing new therapies for breast cancer.

Cellular senescence irreversibly arrests cell proliferation, accompanied by a multi-component senescence-associated secretory phenotype (SASP) that participates in several age-related diseases. Using stable isotope labeling with amino acids in cell culture (SILAC), we identify 343 SASP proteins that senescent human fibroblasts secrete at 2-fold or higher levels compared to quiescent cell counterparts. Bioinformatic analysis revealed that 44 of these SASP factors are involved in hemostasis, a process not previously linked with cellular senescence. We validate the expression of some of these SASP factors in cell culture and in vivo. Mice treated with the chemotherapeutic agent doxorubicin, which induces widespread cellular senescence in vivo, show increased blood clotting. Conversely, use of a transgene that permits the selective removal of senescent cells shows that senescent cell clearance attenuates the increased clotting shown by doxorubicin-treated mice. Together, our study characterizes a SASP profile using SILAC, unveiling a novel function for cellular senescence in hemostasis.

Contains fulltext : 174435.pdf (Publisher’s version ) (Open Access) Cellular senescence is defined as an irreversible growth arrest with the acquisition of a distinctive secretome. The growth arrest is a potent anticancer mechanism whereas the secretome facilitates wound healing, tissue repair, and development. The senescence response has also become increasingly recognized as an important contributor to aging and age-related diseases, including cancer. Although oncogenic mutations are capable of inducing a beneficial senescence response that prevents the growth of premalignant cells and promotes cancer immune-surveillance, the secretome of senescent cells also includes factors with pro-tumorigenic properties. On June 23rd and 24th, 2016, the Division of Cancer Biology of the National Cancer Institute sponsored a workshop to discuss the complex role of cellular senescence in tumorigenesis with the goal to define the major challenges and opportunities within this important field of cancer research. Additionally, it was noted how the development of novel tools and technologies are required to accelerate research into a mechanistic understanding of senescent cells in carcinogenesis in order to overcome the current limitations in this exciting, yet ill-defined area.

© 2016 Cancer Research UK. Cellular senescence is an established tumour-suppressive mechanism that prevents the proliferation of premalignant cells. However, several lines of evidence show that senescent cells, which often persist in vivo, can also promote tumour progression in addition to other age-related pathologies via the senescence-associated secretory phenotype (SASP). Moreover, new insights suggest the SASP can facilitate tissue repair. Here, we review the beneficial and detrimental roles of senescent cells, highlighting conditions under which the senescence response does and does not promote pathology, particularly cancer. By better understanding the context-dependent effects of cellular senescence, it may be feasible to limit its detrimental properties while preserving its beneficial effects, and develop novel therapeutic strategies to prevent or treat cancer and possibly other age-associated diseases.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687-694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER- patients.

Squamous cell carcinoma (SCC) is the second most common non-melanoma skin cancer, and has a high rate of recurrence and metastasis. Systemic genotoxic chemotherapeutic drugs such as doxorubicin (Doxo) can be used to treat SCC, but these agents can have deleterious long-term side effects, including fueling the development of more aggressive cancers. Some of the adverse effects of genotoxic chemotherapies might be due to their ability to induce cellular senescence. Cellular senescence is a tumor suppressive mechanism that entails a permanent cell growth arrest accompanied by several metabolic changes. Senescent cells increase during aging, and accumulating evidence suggests they can contribute to a variety of age related pathologies, including cancer, by secreting numerous pro-inflammatory molecules, growth factors and proteases, a feature termed the senescence-associated secretory phenotype (SASP). We recently showed that genotoxic and cytotoxic chemotherapeutic agents induce senescence and a SASP in vivo. However, a direct role for senescent cells in skin carcinogenesis in vivo remains unexplored. To investigate the role of senescent cells and the SASP in squamous cell skin carcinogenesis, we first induced senescence with Doxo in p16-3MR transgenic mice. These mice are designed to permit the selective elimination of senescent cells expressing the established senescence marker p16INK4a (p16) by administrating ganciclovir (GCV) which has a high affinity for the herpes simplex viral thymidine kinase (HSV-TK). Phosphorylated GCV is a DNA chain terminator that kills HSV-TK expressing cells by apoptosis. After Doxo treatment, we initiated skin SCC in p16-3MR mice with 7, 12-dimethylbenz[a]anthracene (DMBA), followed by treatment with 12-O-Tetradecanoylphorbol-13-acetate (TPA) to promote skin carcinogenesis. 25 weeks after Doxo treatment, we observed a significant increase in p16 mRNA levels as well as an increase in expression of the SASP factors IL-1α, MMP-9, and IL-17 in the skin of Doxo- compared to PBS- treated mice. Further, the presence of senescent cells resulted in significantly larger and more proliferative tumors compared to PBS-treated mice. Importantly, systemic elimination of Doxo-induced senescent cells using GCV reduced the expression of SASP factors and significantly reduced tumor size, suggesting that senescent cells fuel skin tumor progression. These findings will help our understanding of the role of chemotherapy-induced cellular senescence in skin carcinogenesis and will pave the way for developing novel therapeutics against squamous cell carcinoma and the deleterious side effects of genotoxic chemotherapies. Citation Format: Fatouma Alimirah, Alexis Valdovinos, Emily Chang, Sena Alptekin, Tanya Pulido, Elijah Jones, Chandani Limbad, Michael Velarde, Marco Demaria, Albert Davalos, Judith Campisi. Cellular senescence drives skin carcinogenesis [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 466.

It is well-established that CYP24, an immediate target gene of VDR is upregulated by VDR ligands. This study is focused on the functional role of unliganded VDR by investigating the correlation between the expression of VDR protein and basal mRNA levels of CYP24 in breast cancer cell lines. Analyses of multiple breast cancer cell lines demonstrated an inverse correlation between VDR protein expression and CYP24 mRNA expression levels; while in the presence of ligand, VDR protein level was positively correlated with CYP24 expression. In MCF-7 cells, VDR was mainly distributed in the nuclei in the absence of ligand. VDR overexpression in MCF-7 cells and MDA-MB231 cells decreased CYP24 mRNA expression levels and CYP24 promoter activity. Conversely, knock-down of VDR using siRNA techniques in MCF-7 and T47D cells significantly increased CYP24 mRNA expression. We also found that overexpression of VDR with a polymorphic site (FokI-FF) at its AF-1 domain, which makes VDR shorter by three amino acids, failed to repress CYP24 promoter activity. This report provides conclusive evidence for the repressive action of unliganded VDR on the expression of its target gene CYP24 and the importance of an intact VDR AF-1 domain for its repressive action.

Cancer related deaths in breast cancer patients are due to metastasis of the disease. Murine 4T1 cells (Murine mammary cancer cell line developed from 6-thioguanine resistant tumor) provide an excellent research tool for metastasis related studies because these cells are highly aggressive and readily metastasize to the lungs. In this study we determined the effect of Deguelin on in vivo/vitro growth and metastasis of 4T1 cells. Deguelin inhibited the in vitro growth of 4T1 cells in a time and dose dependent manner accompanied with reduced nuclear PCNA immunostaining. In cells treated with Deguelin, reduced expression of nuclear c-Met, and its downstream targets such p-ERK and p-AKT was observed. Deguelin reduced the cell migration in 4T1 cells as determined by scratch wound assay. Combined treatment with Deguelin + ERK or PI3K/AKT inhibitor had no additional effect on cell migration. These results indicated that the action of Deguelin on cell migration may be mediated by AKT and ERK mediated signaling pathways. In vivo, Deguelin treatment significantly inhibited growth of 4T1 cells. Deguelin also reduced the occurrence of metastatic lung lesions by 33 % when cells were injected intravenously into Balb/c female mice. There was no difference in the body weight, nor was there a difference in liver and spleen weights between vehicle treated-control and Deguelin-treated animals, which indicated that Deguelin was nontoxic at the dose used in the present study. These results provide rationale for developing Deguelin as a chemotherapeutic agent for triple negative breast cancer patients.

The benefit of vitamin D in cancer prevention and to certain extent therapy has been well recognized. The active form of vitamin D, 1,25-dihydroxycholecalciferol (1,25(OH)2 D3) is a natural ligand for vitamin D receptor (VDR). Since 1,25(OH)2D3 exerts toxic effects at a concentration that is beneficial, nearly 1500 analogs of vitamin D have been synthesized and evaluated for their efficacy in a variety of carcinogenesis and human cancer models both in vitro and in vivo. Among these only a handful of them have been approved for evaluation in clinical trials for leukemia, breast, prostate and colon cancers. The mechanism of vitamin D action is mediated by the nuclear VDR and the signaling cascade for its action is extensively reported. In this review we focus on the newer concepts for vitamin D action. These include (1) differential effects of vitamin D in maintaining cell proliferation when the cells are under stress but suppressing cell growth when the cells are transformed; (2) functional significance of VDR polymorphism in potential vitamin D responsiveness; (3) regulation of constitutive splicing of vitamin D target gene, CYP24a, by the hormone and its significance; and (4) regulation of microRNA by vitamin D in breast cancer. It is anticipated that the new work in these selective areas would expand the understanding of vitamin D in breast cancer prevention and therapy.

Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ERα) physically binds to peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits its transcriptional activity. The interaction between PPARγ and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPARγ and VDR signaling, and for the first time we show that PPARγ physically associates with VDR in human breast cancer cells. We found that overexpression of PPARγ decreased 1α,25-dihydroxyvitamin D(3) (1,25D(3)) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPARγ's hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγ's AF2 domain attenuated its repressive action on 1,25D(3) transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPARγ was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXRα). Overexpression of RXRα blocked PPARγ's suppressive effect on 1,25D(3) action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways.

Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10 μM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's 't' test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50-60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs alone. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PR-B/EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients.

25-Hydroxyvitamin D(3) (25(OH)D(3)) is a prohormone and a major vitamin D metabolite. The discovery of (25(OH)D(3)) 1 alpha-hydroxylase in many vitamin D target organs has yielded an increased interest in defining the role(s) of 25(OH)D(3) in these tissues. The etiology of cancer appears to be complex and multi-factorial. Cellular stress (e.g., DNA damage, hypoxia, oncogene activation) has been identified as one of the key factors responsible for initiating the carcinogenesis process. In this study, we investigated whether 25(OH)D(3) protects breast epithelial cells from cellular stress using an established breast epithelial cell line MCF12F. To better elucidate the role of 25(OH)D(3) in the stress response, we used multiple in vitro stress models including serum starvation, hypoxia, oxidative stress, and apoptosis induction. Under all these stress conditions, 25(OH)D(3) (250 nmol/L) treatment significantly protected cells against cell death. Low-serum stress induced p53 expression accompanied with downregulation of PCNA, the presence of 25(OH)D(3) consistently inhibited the alteration of p53 and PCNA, suggesting that these molecules were involved in the stress process and may be potential target genes of 25(OH)D(3). miRNA microarray analysis demonstrated that stress induced by serum starvation caused significant alteration in the expression of multiple miRNAs including miR182, but the presence of 25(OH)D(3) effectively reversed this alteration. These data suggest that there is a significant protective role for 25(OH)D(3) against cellular stress in the breast epithelial cells and these effects may be mediated by altered miRNA expression.

Background Increased levels of interferon (IFN)-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22) in human normal prostate epithelial cells and diploid fibroblasts (HDFs) are associated with the onset of cellular senescence. However, the molecular mechanisms by which the IFI16 protein contributes to cellular senescence-associated cell growth arrest remain to be elucidated. Here, we report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT) gene. Methodology/Principal Findings We optimized conditions for real-time PCR, immunoblotting, and telomere repeat amplification protocol (TRAP) assays to detect relatively low levels of hTERT mRNA, protein, and telomerase activity that are found in HDFs. Using the optimized conditions, we report that treatment of HDFs with inhibitors of cell cycle progression, such as aphidicolin or CGK1026, which resulted in reduced steady-state levels of IFI16 mRNA and protein, was associated with increases in hTERT mRNA and protein levels and telomerase activity. In contrast, knockdown of IFI16 expression in cells increased the expression of c-Myc, a positive regulator of hTERT expression. Additionally, over-expression of IFI16 protein in cells inhibited the c-Myc-mediated stimulation of the activity of hTERT-luc-reporter and reduced the steady-state levels of c-Myc and hTERT. Conclusions/Significance These data demonstrated that increased levels of IFI16 protein in HDFs down-regulate the expression of hTERT gene. Our observations will serve basis to understand how increased cellular levels of the IFI16 protein may contribute to certain aging-dependent diseases.

IFN-inducible IFI16 protein (encoded by IFI16 gene at 1q23.1) is the human member of the IFN-inducible structurally related p200 family proteins. Increased expression of the IFI16 protein, a positive modulator of p53-mediated transcription, in normal old human diploid fibroblasts (HDF) is associated with cellular senescence-mediated cell growth arrest. However, the underlying mechanisms that contribute to transcriptional activation of the IFI16 gene in old HDFs remain to be elucidated. Here, we reported that functional activation of p53 in normal young HDFs and p53-null Saos2 cell line resulted in transcriptional activation of the IFI16 gene. We identified a potential p53 DNA-binding site (indicated as IFI16-p53-BS) in the 5′-regulatory region of the IFI16 gene. Importantly, p53 bound to IFI16-p53-BS in a sequence-specific manner in gel-mobility shift assays. Furthermore, p53 associated with the 5′-regulatory region of the IFI16 gene in chromatin immunoprecipitation assays. Interestingly, p53 associated with the regulatory region of the IFI16 gene only on treatment of cells with DNA-damaging agents or in the old, but not in the young, HDFs. Importantly, our promoter-reporter assays, which were coupled with site-directed mutagenesis of IFI16-p53-BS, showed that p53 activates transcription of the IFI16 gene in HDFs through the p53 DNA-binding site. Together, our observations provide support for the idea that up-regulation of IFI16 expression by p53 and functional interactions between IFI16 protein and p53 contribute to cellular senescence. (Mol Cancer Res 2008;6(11):1732–41)

Increased expression of IFI16 protein (encoded by the IFI16 gene) in normal human prostate epithelial cells is associated with cellular senescence-associated cell growth arrest. Consistent with a role for IFI16 protein in cellular senescence, the expression of IFI16 protein is either very low or not detectable in human prostate cancer cell lines. We now report that treatment of DU-145 and LNCaP prostate cancer cell lines with histone deacetylase inhibitor trichostatin A (TSA) or CGK1026 resulted in transcriptional activation of the IFI16 gene. The induction of IFI16 protein in LNCaP cells was dependent on the duration of TSA treatment. Furthermore, TSA treatment of LNCaP cells up-regulated the expression of Janus-activated kinase 1 protein kinase and modulated the transcription of certain IFN-activatable genes. However, overexpression of exogenous Janus-activated kinase 1 protein in LNCaP cells and treatment of cells with IFNs (α and γ) did not increase the expression of IFI16. Instead, the transcriptional activation of IFI16 gene by TSA treatment of LNCaP cells was dependent on transcriptional activation by c-Jun/activator protein-1 transcription factor. Importantly, increased expression of IFI16 in LNCaP cells was associated with decreases in the expression of androgen receptor and apoptosis of cells. Conversely, knockdown of IFI16 expression in TSA-treated LNCaP cells increased androgen receptor protein levels with concomitant decreases in apoptosis. Together, our observations provide support for the idea that histone deacetylase–dependent transcriptional silencing of the IFI16 gene in prostate epithelial cells contributes to the development of prostate cancer. (Mol Cancer Res 2007;5(3):251–9)

The majority of human prostate cancer cell lines, including the two “classical” cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299–315), but not the variant N- or C-terminal region (amino acids 1–20 and 900–919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21CIP1 protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.

Proliferation of normal and malignant prostate epithelium is regulated by androgen stimulation via both the androgen receptor (AR)-positive stromal and epithelial cells. However, it is not known how AR expression is regulated in human prostate cells. We report that treatment of normal human prostate stromal cells (PrSCs) with type I IFN (alpha or beta), but not type II IFN (gamma), resulted in increased levels of AR protein. The maximal increase in AR protein levels was dependent on the dose and the duration of the IFN-alpha treatment. We found that the increase in AR protein levels was independent of de novo transcription and protein synthesis. Interestingly, the IFN-alpha treatment of PrSCs resulted in considerable nuclear accumulation of AR, stimulation of AR-mediated transcription of reporter genes, and retardation of cell proliferation. Furthermore, treatment of normal human prostate epithelial cells with IFNs (alpha, beta or gamma) also resulted in increased levels of AR protein. Together, our observations identify the androgen receptor as an IFN-regulated protein in normal human prostate stromal and epithelial cells and predict that IFN-induced levels of AR in prostate cells contribute to the regulation of androgen signaling.

The estrogen receptor alpha (ERα), a nuclear receptor and transcription factor governs the action of estrogen. This hormone is the major contributor to normal breast development and breast carcinogenesis. Thus, antiestrogens are widely utilized to treat and circumvent the progression of hormone receptor positive breast cancer. However, resistance to these agents develops after sustained use and major risk factors such as enhanced susceptibility to endometrial cancer have been associated with this type of treatment. In an effort to develop natural therapeutic compounds against ERα positive breast cancer, the effect of deguelin, a rotenoid isolated from an African plant; Mundulea sericea on ERα positive human breast cancer cells was investigated. Here we report that deguelin treatment downregulated the expression of ERα protein in a dose dependent manner in MCF-7 and T47D human breast cancer cell lines as determined by immunoblotting. Quantitative RT-PCR analysis revealed that the mRNA expression of ERα target genes Cathepsin D (CTSD), Trefoil factor 1 (TFF1) and the Progesterone receptor (PGR) was also downregulated by 50% in the presence of deguelin in MCF-7 cells. Furthermore, deguelin treatment significantly inhibited the proliferation of these two cell lines. Importantly, estrogen mediated stimulation of cell growth was reduced by 30% in MCF-7 cells after deguelin treatment. Together these findings suggest that deguelin suppresses ERα signaling, paving the way for the development of a novel natural therapeutic agent against endocrine receptor positive breast cancer. Future studies are aimed at deciphering the molecular mechanisms by which deguelin adversely affects ERα function. (This work was supported by NCI CA140321). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 573. doi:1538-7445.AM2012-573

Human breast cancers overexpressing HER-2 are generally highly aggressive. Current therapeutic strategies to target HER-2 signaling pathway with antibodies or small molecule tyrosine kinase inhibitors have shown impressive success in the management of these tumor types, although most women exhibit short term response to these therapies. Acquired resistance to HER-2 target drugs, especially Herceptin is a major challenge to clinicians. Patients receiving Herceptin therapy invariably recur and most often with highly aggressive tumors. Thus identifying new drugs which target multiple molecular pathways is desired to treat Herceptin resistant breast cancer. Using paired HER-2 overexpressing parental Herceptin responsive (BT-474) and its Herceptin resistant variant cells (BT-474HR) we showed that acquired resistance to Herceptin is associated with enhanced expression of HER-2, EGFR, CyclinD1, P-AKT (Ser-473), PI-3K, JAB1 proteins and decreased expression of nuclear pten and p27 proteins. These results are similar to those observed in recurrent breast tumors in patients following Herceptin treatment. Deguelin, a natural product derivative of rotenoids and has been shown to inhibit growth of diverse cancer types. Deguelin is a known AKT inhibitor, however its efficacy is never tested in breast cancers resistant to Herceptin which express high levels of p-AKT. We examined effect of Deguelin on HER-2 overexpressing BT-474 BT-474-HRcells. Deguelin inhibited growth of both, BT-474 and BT-474HR cells in concentration (0-250nM) and time dependent (24-72h) manner. There was a 40-50%- inhibition of both BT474and BT-474HR cells at 25nM Deguelin treatment at 72h. Deguelin decreased cell surface expression of HER-2 in both these cell lines. In parental BT-474 cells both Herceptin 5-10ug/ml and Deguelin down regulated HER-2 expression, but only Deguelin reduced HER-2 (125-250nM) over expression in both BT-474 and BT-474HR cells. Down regulation of HER-2 following Deguelin treatment was associated with simultaneous down regulation of nuclear accumulation of cyclin D1, and JAB1, and p-AKT (Ser-473) proteins and up regulation of nuclear p27 and p53in both BT-474 and BT474 HR cells. These results suggest that Deguelin inhibits growth of both HER-2 overexpressing and those resistant to Herceptin therapy breast cancer cells. Effect of Deguelin appears to be mediated through down regulation of HER-2, PI3K AKT, JAB1, Cyclin D1, and p27 pathway. Our preliminary results warrant further investigation into Deguelin as a potential therapeutic agent for Herceptin resistant breast cancers. (This work was supported by NCI CA140321.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3812. doi:1538-7445.AM2012-3812

Colon cancer cells interact with immune cells in vivo, creating a microenvironment which affects both tumor growth and treatment. Macrophages are often the most abundant immune cells in the tumor microenvironment and are involved in inflammation. Since inflammation is tightly associated with colon carcinogenesis, it is important to understand how vitamin D affects the interaction between colon epithelial cells and macrophages. In this study, we used a co-culture system to examine the role of 25-hydroxyvitamin D3 (25(OH)D3) on the clonogenic growth of colon cancer cells and on the profiling of cytokines/chemokines. 25(OH)D3 at 250 nM had minor effect on clonogenic growth in CaCo-2 cells, co-culture of macrophage-like THP-1 cells with CaCo-2 cells significantly stimulated the clonogenic growth of CaCo-2 cells in the absence of 25(OH)D3. However, in the presence of 25(OH)D3, THP-1 cells failed to stimulate the the clonogenic growth of CaCo-2 cells. Cytokine antibody array demonstrated that several cytokines were differentially expressed in the medium after co-culture of the two cell lines for 2 days in the absence of 25(OH)D3. These include increased expression of GROα, MCP-1 and decreased expression of TNFα, Angiogenin; however, in the presence of 25(OH)D3, MCP-2 and MDC were significantly induced and TNFα level was restored. Proliferation assay showed that MCP-2 and MDC had minor or no effect on cell proliferation in CaCo-2 cells, suggesting that these chemokines mainly target macrophages. 25(OH)D3 treatment of THP-1 cells for 24h also induced CYP24, an immediate vitamin D target gene and enhanced MDC and MCP-2 expression as evaluated by qRT-PCR. These results demonstrate that 25(OH)D3 at 250 nM might selectively target macrophages in a co-culture system with colon cells and block the interaction between colon cancer cells and macrophages, which may result in prevention of inflammation. [Supported by NCI R01 CA121157]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 456. doi:10.1158/1538-7445.AM2011-456

Previous studies have demonstrated a positive interaction between vitamin D and peroxisome proliferator- activated receptor γ (PPARγ) mediated signaling while other studies have shown an inverse relationship between these two pathways. Therefore, we sought to determine the functional interaction between the vitamin D receptor (VDR) and PPARγ in human breast cancer cells. We report the physical interaction and nuclear colocalization of VDR and PPARγ in the T47D human breast cancer cell line as determined by co-immunoprecipitation and immunofluorescence respectively. Furthermore, 1alpha,25-dihydroxyvitamin D3 (1,25D3) treatment of MCF-7 and T47D cells significantly upregulated PPARγ protein expression in a dose and time dependent manner. Conversely, knockdown of unliganded VDR also upregulated PPARγ protein expression, suggesting that it is a potential VDR target gene. Thus, VDR itself in the absence of ligand, can suppress PPARγ basal expression. This paradoxical effect may be due to the differential binding of VDR to its coactivators or corepressors during the liganded and unliganded state. Consistent with this, the basal expression of PPARγ mRNA was inversely correlated with VDR mRNA expression in several human breast cancer cell lines. Reciprocally, a PPARγ agonist attenuated vitamin D mediated upregulation of vitamin D target genes CYP24 and p21. Similarly, the activity of VDRE, a vitamin D responsive reporter was decreased by 2 fold in the presence of the PPARγ agonist. Together, these observations infer that VDR and PPARγ protein interactions negatively influence their respective pathways. (This work was supported by NCI predoctoral fellowship 1F31CA132619 (F.A.) and NCI R03 CA121365 (X.P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5718.