G1 Growth Arrest Research Articles

Abstract 5709: PF-07104091, a first-in-class CDK2-selective inhibitor for the treatment of HR+/HER2- breast cancer and CCNE1high ovarian cancer

Abstract The selective inhibition of Cyclin\CDK complexes controlling cell cycle progression has been established as cancer therapy by selective CDK4/6 inhibitors in HR+/HER2- breast cancer. Expanding control of the cell cycle through selective inhibition of CDK2 offers novel therapeutic opportunities in cancer, including targeting CCNE1 amplified tumors and countering resistance to CDK4\6 inhibitors in ER+ breast cancer. PF-07104091 is a first-in-class CDK2-selective inhibitor under clinical investigation in patients with HR+/HER2- breast cancer and ovarian cancer. Preclinical studies using PF-07104091 establish the therapeutic impact of CDK2 inhibition in both diseases and highlight distinct mechanistic roles for CDK2 in control of cancer cell proliferation. In models of CCNE1 amplified ovarian cancer, CDK2 plays the dominant role in control of RB1 phosphorylation and the G1 checkpoint. CDK2 inhibition with PF-07104091 induces G1 growth arrest and controls tumor xenograft growth as single agent therapy. In ER+ breast models CDK2 plays a supportive role in the control of RB1 phosphorylation cooperating with CDK4\6. Whole genome CRISPR KO and CRISPR activation screens in conjunction with CDK4\6 inhibition establish CDK2 KO as a primary sensitizer to CDK4\6 inhibition, and support Cyclin E\CDK2 complexes as the driver of resistance to CDK4\6 inhibitors in ER+ breast models. PF-07104091 combined with CDK4\6 inhibitor Palbociclib or CDK4-selective inhibitor PF-0060 synergistically controls proliferation of ER+ BC cells in vitro and induces tumor regression in ER+ BC xenograft models, including PDX models with acquired resistance to CDK4\6 inhibitors and endocrine therapy. Citation Format: Chen Shen, Ming Qiu, Nanni Huser, Bernadette Pascual, Qin Zhang, Todd VanArsdale, Anwar Murtaza, Jonathan Almaden, berly Kim, Lars Anders, Koleen Eisele. PF-07104091, a first-in-class CDK2-selective inhibitor for the treatment of HR+/HER2- breast cancer and CCNE1high ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5709.

VRK3 depletion induces cell cycle arrest and metabolic reprogramming of pontine diffuse midline glioma - H3K27 altered cells.

We previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-H3K27M cell fitness. Gene expression studies after VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knockdown. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201.

Androgen Receptor Activation Induces Senescence in Thyroid Cancer Cells.

Thyroid cancer (TC) is the most common endocrine malignancy, with an approximately three-fold higher incidence in women. TCGA data indicate that androgen receptor (AR) RNA is significantly downregulated in PTC. In this study, AR-expressing 8505C (anaplastic TC) (84E7) and K1 (papillary TC) cells experienced an 80% decrease in proliferation over 6 days of exposure to physiological levels of 5α-dihydrotestosterone (DHT). In 84E7, continuous AR activation resulted in G1 growth arrest, accompanied by a flattened, vacuolized cell morphology, with enlargement of the cell and the nuclear area, which is indicative of senescence; this was substantiated by an increase in senescence-associated β-galactosidase activity, total RNA and protein content, and reactive oxygen species. Additionally, the expression of tumor suppressor proteins p16, p21, and p27 was significantly increased. A non-inflammatory senescence-associated secretory profile was induced, significantly decreasing inflammatory cytokines and chemokines such as IL-6, IL-8, TNF, RANTES, and MCP-1; this is consistent with the lower incidence of thyroid inflammation and cancer in men. Migration increased six-fold, which is consistent with the clinical observation of increased lymph node metastasis in men. Proteolytic invasion potential was not significantly altered, which is consistent with unchanged MMP/TIMP expression. Our studies provide evidence that the induction of senescence is a novel function of AR activation in thyroid cancer cells, and may underlie the protective role of AR activation in the decreased incidence of TC in men.

Type 2 deiodinase is expressed in anaplastic thyroid carcinoma and its inhibition causes cell senescence.

Anaplastic thyroid cancer (ATC) is a rare thyroid tumor that frequently originates from the dedifferentiation of a well-differentiated papillary or follicular thyroid cancer. Type 2 deiodinase (D2), responsible for the activation of the thyroid hormone thyroxine into tri-iodothyronine (T3), is expressed in normal thyroid cells and its expression is strongly downregulated in papillary thyroid cancer. In skin cancer, D2 has been associated with cancer progression, dedifferentiation, and epithelial-mesenchymal transition. Here, we show that D2 is highly expressed in anaplastic compared to papillary thyroid cancer cell lines and that D2-derived T3 is required for ATC cell proliferation. D2 inhibition is associated with G1 growth arrest and induction of cell senescence, together with reduced cell migration and invasive potential. Finally, we found that mutated p5372R(R248W), frequently found in ATC, is able to induce D2 expression in transfected papillary thyroid cancer cells. Our results show that the action of D2 is crucial for ATC proliferation and invasiveness, providing a potential new therapeutic target for the treatment of ATC.

The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo.

Treatment of rhabdomyosarcoma (RMS), the most common a soft tissue sarcoma in childhood, provides intensive multimodal therapy, with radiotherapy (RT) playing a critical role for local tumor control. However, since RMS efficiently activates mechanisms of resistance to therapies, despite improvements, the prognosis remains still largely unsatisfactory, mainly in RMS expressing chimeric oncoproteins PAX3/PAX7-FOXO1, and fusion-positive (FP)-RMS. Cardiac glycosides (CGs), plant-derived steroid-like compounds with a selective inhibitory activity of the Na+/K+-ATPase pump (NKA), have shown antitumor and radio-sensitizing properties. Herein, the therapeutic properties of PBI-05204, an extract from Nerium oleander containing the CG oleandrin already studied in phase I and II clinical trials for cancer patients, were investigated, in vitro and in vivo, against FN- and FP-RMS cancer models. PBI-05204 induced growth arrest in a concentration dependent manner, with FP-RMS being more sensitive than FN-RMS, by differently regulating cell cycle regulators and commonly upregulating cell cycle inhibitors p21Waf1/Cip1 and p27Cip1/Kip1. Furthermore, PBI-05204 concomitantly induced cell death on both RMS types and senescence in FN-RMS. Notably, PBI-05204 counteracted in vitro migration and invasion abilities and suppressed the formation of spheroids enriched in CD133+ cancer stem cells (CSCs). PBI-05204 sensitized both cell types to RT by improving the ability of RT to induce G2 growth arrest and counteracting the RT-induced activation of both Non-Homologous End-Joining and homologous recombination DSBs repair pathways. Finally, the antitumor and radio-sensitizing proprieties of PBI-05204 were confirmed in vivo. Notably, both in vitro and in vivo evidence confirmed the higher sensitivity to PBI-05204 of FP-RMS. Thus, PBI-05204 represents a valid radio-sensitizing agent for the treatment of RMS, including the intrinsically radio-resistant FP-RMS.

MS-275 (Entinostat) Promotes Radio-Sensitivity in PAX3-FOXO1 Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2, SOD, CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

The inhibitory effect of canine interferon gamma on the growth of canine tumors

Recombinant canine interferon-γ (rc-IFNγ; InterdogⓇ) was exclusively approved as a therapeutic for canine atopic dermatitis. However, it has been used off-label for the treatment of canine cancer. We examined the inhibitory effect of rc-IFNγ on the growth of canine tumor cell lines and analyzed its mechanism of action. Three (CTB-p, CTB-m, and CNM-m) out of seven mammary gland tumor cell lines and two (VIMC and CoMS) out of four mast cell tumor cell lines showed remarkable growth inhibition after treatment with rc-IFNγ. However, one (CLBL-1) out of nine lymphoma cell lines showed a significant amount of cell death. Using CTB-p and CTB-m cell lines, we showed that STAT1 was essential for inducing the growth inhibitory effect of rc-IFNγ. Although rc-IFNγ induced G1 growth arrest in CTB-p cell line, treatment with rc-IFNγ did not alter the expression of cell cycle regulatory proteins. In this study, we observed direct cytotoxicity or cytostatic effects of rc-IFNγ in canine tumor cell lines. However, the detailed mechanisms responsible for these effects need to be elucidated in the future.

Abstract 1558: Therapeutic targeting of cell surface plectin induces anti-cancer immune response and pancreatic cancer regression

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) is the 3rd deadliest cancer, diagnosed typically in advanced stages, with only an 8% 5-year survival rate, thus demonstrating the need for novel therapeutic approaches that significantly enhance chemo- and/or immune-therapy. Our team previously identified a promising functional target for cancer therapy in PDA, cell surface plectin 1 (CSP1) that is aberrantly expressed on PDA cells and thus a cell surface-associated biomarker of cancer. CSP1 expression first becomes apparent in high grade dysplasias, remaining high in early and advanced cancers and in metastases. Our first-in-human imaging trial in PDA patients using a CSP1-targeted imaging agent revealed that CSP is an available target and accessible for binding, a potentially a target for cancer therapy. We hypothesized that a monoclonal antibody (mAb) against CSP1 could lead to novel pancreatic cancer treatment options, thus, we developed a therapeutic mAb, e.g., ZB131, representing a first-in-class antibody selectively targeting CSP1. Methods: ZB131 is a humanized mAb targeted against human plectin 1 (rhSec8) that also binds murine CSP1. ZB131 affinity and its effect on cancer cells including proliferation, cytotoxicity, and migration were tested in vitro using saturation binding, SRB-based survival assays, flow cytometry, and migration assays on various pancreatic cell types and homeostatic “normal” controls. In vivo validation was performed using two nu/nu mouse models bearing subcutaneous MiaPACA2 or Yapc PDA cells, and a syngeneic KPC-derived tumor model to also evaluate immune responses to tumors treated with ZB131 or IgG control. Results: ZB131 exhibits high specificity and high affinity (0.4±0.1nM) to CSP1, and functionally induces G0 growth arrest followed by necrotic cell death of PDA cells in culture, and is synergistic with gemcitabine resulting in a 50-fold decrease in IC50. In vivo, in subcutaneous xenograft models, ZB131 monotherapy decreased PDA tumor volume 5-fold as compared to controls, and in combination with cisplatin resulted in sustained tumor reduction with greater than 85% tumor necrosis. In subcutaneous syngeneic PDA models, ZB131 induced complete tumor regression within 35 days mediated by an anti-tumor immune response as upon tumor rechallenge, full tumor regression was again achieved without additional ZB131 therapy. Leukocyte complexity analysis of regressing PDA tumors versus controls revealed an ~3-fold increase in effector and central memory T cells. Conclusion: CSP1 is a first in class anti-cancer target expressed on the cell surface of PDA, as well as other cancers including ovarian, esophageal and head neck. ZB131, an anti-CSP1 mAb, induces tumor cell intrinsic cell death, as well as a robust anti-tumor T cell response leading to complete tumor regression indicating the potential therapeutic efficacy of ZB131 in late-stage cancers. Citation Format: Julien Dimastromatteo, Amanda Poisonnier, Samantha Perez, Lisa Coussens, Kimberly Kelly. Therapeutic targeting of cell surface plectin induces anti-cancer immune response and pancreatic cancer regression [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 1558.

Intracellular vesicle trafficking plays an essential role in mitochondrial quality control.

The Drosophila gene products Bet1, Slh, and CG10144, predicted to function in intracellular vesicle trafficking, were previously found to be essential for mitochondrial nucleoid maintenance. Here we show that Slh and Bet1 cooperate to maintain mitochondrial functions. In their absence, mitochondrial content, membrane potential, and respiration became abnormal, accompanied by mitochondrial proteotoxic stress, but without direct effects on mtDNA. Immunocytochemistry showed that both Slh and Bet1 are localized at the Golgi, together with a proportion of Rab5-positive vesicles. Some Bet1, as well as a tiny amount of Slh, cofractionated with highly purified mitochondria, while live-cell imaging showed coincidence of fluorescently tagged Bet1 with most Lysotracker-positive and a small proportion of Mitotracker-positive structures. This three-way association was disrupted in cells knocked down for Slh, although colocalized lysosomal and mitochondrial signals were still seen. Neither Slh nor Bet1 was required for global mitophagy or endocytosis, but prolonged Slh knockdown resulted in G2 growth arrest, with increased cell diameter. These effects were shared with knockdown of betaCOP but not of CG1044, Snap24, or Syntaxin6. Our findings implicate vesicle sorting at the cis-Golgi in mitochondrial quality control.

Role of 1α,25-Dihydroxyvitamin D3 in Adipogenesis of SGBS Cells: New Insights into Human Preadipocyte Proliferation

Background/Aims: Compared with non-obese individuals, obese individuals commonly store more vitamin D in adipose tissue. VDR expression in adipose tissue can influence adipogenesis and is therefore a target pathway deserving further study. This study aims to assess the role of 1,25(OH)₂D₃ in human preadipocyte proliferation and differentiation. Methods: RTCA, MTT, and trypan blue assays were used to assess the effects of 1,25(OH)₂D₃ on the viability, proliferation, and adipogenic differentiation of SGBS cells. Cell cycle and apoptosis analyses were performed with flow cytometry, triglycerides were quantified, and RT-qPCR was used to assess gene expression. Results: We confirmed that the SGBS cell model is suitable for studying adipogenesis and demonstrated that the differentiation protocol induces cell maturation, thereby increasing the lipid content of cells independently of treatment. 1,25(OH)₂D₃ treatment had different effects according to the cell stage, indicating different modes of action driving proliferation and differentiation. In preadipocytes, 1,25(OH)₂D₃ induced G1 growth arrest at both tested concentrations without altering CDKN1A gene expression. Treatment with 100 nM 1,25(OH)₂D₃ also decreased MTT absorbance and the lipid concentration. Moreover, increased normalized cell index values and decreased metabolic activity were not induced by proliferation or apoptosis. Exposure to 100 nM 1,25(OH)₂D₃ induced VDR, CEBPA, and CEBPB expression, even in the preadipocyte stage. During adipogenesis, 1,25(OH)₂D₃ had limited effects on processes such as VDR and PPARG gene expression, but it upregulated CEBPA expression. Conclusions: We demonstrated for the first time that 1,25(OH)₂D₃ induces changes in preadipocytes, including VDR expression and growth arrest, and increases the lipid content in adipocytes treated for 16 days. Preadipocytes are important cells in adipose tissue homeostasis, and understanding the role of 1,25(OH)₂D₃ in adipogenesis is a crucial step in ensuring adequate vitamin D supplementation, especially for obese individuals.

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype, induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

BackgroundEPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.MethodsEPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.ResultsOur study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.ConclusionsTaken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

The anti-aging promise of p21

KEYWORDS: agingapoptosiscellular senescenceDNA damage response (DDR)extra cellular matrix (ECM)fibrosisJNKp21(CDKN1A)

Abstract 310: Enhanced stability of D-type cyclins correlates with glioblastoma resistance to ionizing radiation

Abstract Transition from G1 to S phase of the cell cycle is promoted by D-type cyclins (D1, D2 and D3), regulatory subunits critical for the activation of cyclin dependent kinases 4 and 6 (CDK4/6), which phosphorylate and inactivate the retinoblastoma protein (pRb) and activate the transcription factor E2F1 to promote the expression of genes essential for DNA synthesis. Deregulated expression of D-type cyclins is frequently observed in human malignancies and correlates with increased proliferation. Cyclin Ds overexpression is also associated with radioresistance, leading to radiotherapy failure and disease recurrence. Cyclin D1 undergoes ubiquitin-dependent degradation following the exposure of cancer cells to ionizing radiation (IR), and this has been shown to be critical for IR-induced G1 growth arrest and sensitivity of cancer cells to IR. While the role of cyclin D1 overexpression in drug resistance is already well documented, less is known about the role of cyclins D2 and D3 in radio-resistance in human cancer cells, particularly in brain neoplasms. In the present study, we investigated the effect of IR on different D-type cyclins (D1, D2, D3) in human glioblastoma cell lines as well as in tumor-derived mouse oligodendrocytes progenitor cells (OPCs). Here, we show that exposure of human astrocytes to increasing levels of IR led to a rapid proteasomal degradation of all three D-type cyclins. On the other hand, IR failed to induce significant cyclin Ds degradation in a number of human glioblastoma cells (U87, A172, U251, Snb19). Analysis of cell cycle profile of all glioblastoma cells tested and exposed to IR showed prominent cell cycle arrest in G1 starting at 24 hours following exposure. Furthermore, we show that the steady-state levels of D-type cyclin is higher in OPCs compared to mouse NIH3T3 cells and fail to undergo proteasomal degradation following IR exposure. Collectively, these results suggest that the degradation of D-type cyclins is not critical for IR-induced G1 cell cycle arrest in GMB and may underlie their resistance to IR. Understanding the role of cyclin Ds in promoting radioresistance may lead to a useful pharmacological strategy to enhance radiotherapy outcome and help implement a more effective treatment modality. Citation Format: Fadila Guessous, Mouadh Benamar, Hui Zhong, Tarek Abbas. Enhanced stability of D-type cyclins correlates with glioblastoma resistance to ionizing radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 310. doi:10.1158/1538-7445.AM2017-310

Inhibition of the PI3K/AKT/mTOR pathway activates autophagy and compensatory Ras/Raf/MEK/ERK signalling in prostate cancer

The PI3K/AKT/mTOR pathway is frequently activated in advanced prostate cancer, due to loss of the tumour suppressor PTEN, and is an important axis for drug development. We have assessed the molecular and functional consequences of pathway blockade by inhibiting AKT and mTOR kinases either in combination or as individual drug treatments. In established prostate cancer cell lines, a decrease in cell viability and in phospho-biomarker expression was observed. Although apoptosis was not induced, a G1 growth arrest was observed in PTEN null LNCaP cells, but not in BPH1 or PC3 cells. In contrast, when the AKT inhibitor AZD7328 was applied to patient-derived prostate cultures that retained expression of PTEN, activation of a compensatory Ras/MEK/ERK pathway was observed. Moreover, whilst autophagy was induced following treatment with AZD7328, cell viability was less affected in the patient-derived cultures than in cell lines. Surprisingly, treatment with a combination of both AZD7328 and two separate MEK1/2 inhibitors further enhanced phosphorylation of ERK1/2 in primary prostate cultures. However, it also induced irreversible growth arrest and senescence.Ex vivo treatment of a patient-derived xenograft (PDX) of prostate cancer with a combination of AZD7328 and the mTOR inhibitor KU-0063794, significantly reduced tumour frequency upon re-engraftment of tumour cells.The results demonstrate that single agent targeting of the PI3K/AKT/mTOR pathway triggers activation of the Ras/MEK/ERK compensatory pathway in near-patient samples. Therefore, blockade of one pathway is insufficient to treat prostate cancer in man.

Establishment and application of a novel patient-derived KIAA1549:BRAF-driven pediatric pilocytic astrocytoma model for preclinical drug testing.

Pilocytic astrocytoma (PA) is the most frequent pediatric brain tumor. Activation of the MAPK pathway is well established as the oncogenic driver of the disease. It is most frequently caused by KIAA1549:BRAF fusions, and leads to oncogene induced senescence (OIS). OIS is thought to be a major reason for growth arrest of PA cells in vitro and in vivo, preventing establishment of PA cultures. Hence, valid preclinical models are currently very limited, but preclinical testing of new compounds is urgently needed. We transduced the PA short-term culture DKFZ-BT66 derived from the PA of a 2-year old patient with a doxycycline-inducible system coding for Simian Vacuolating Virus 40 Large T Antigen (SV40-TAg). SV40-TAg inhibits TP53/CDKN1A and CDKN2A/RB1, two pathways critical for OIS induction and maintenance. DNA methylation array and KIAA1549:BRAF fusion analysis confirmed pilocytic astrocytoma identity of DKFZ-BT66 cells after establishment. Readouts were analyzed in proliferating as well as senescent states, including cell counts, viability, cell cycle analysis, expression of SV40-Tag, CDKN2A (p16), CDKN1A (p21), and TP53 (p53) protein, and gene-expression profiling. Selected MAPK inhibitors (MAPKi) including clinically available MEK inhibitors (MEKi) were tested in vitro. Expression of SV40-TAg enabled the cells to bypass OIS and to resume proliferation with a mean doubling time of 45h allowing for propagation and long-term culture. Withdrawal of doxycycline led to an immediate decrease of SV40-TAg expression, appearance of senescent morphology, upregulation of CDKI proteins and a subsequent G1 growth arrest in line with the re-induction of senescence. DKFZ-BT66 cells still underwent replicative senescence that was overcome by TERT expression. Testing of a set of MAPKi revealed differential responses in DKFZ-BT66. MEKi efficiently inhibited MAPK signaling at clinically achievable concentrations, while BRAF V600E- and RAF Type II inhibitors showed paradoxical activation. Taken together, we have established the first patient-derived long term expandable PA cell line expressing the KIAA1549:BRAF-fusion suitable for preclinical drug testing.

Abstract 2841: Continuous androgen receptor stimulation in thyroid cancer cells induces irreversible senescence

Abstract Cancer of the thyroid gland accounts for 3.8% of all cancer cases in the United States as indicated by the SEER report by the NCI. The incidence of thyroid cancer has increased three-fold over the last thirty years and the American Cancer Society estimates that there will be approximately 62,450 new cases and 1,950 deaths due to the disease in the country in 2015. Papillary Thyroid Cancer (PTC) is the most common endocrine malignancy with a three-fold higher incidence in women than in men. However, PTC exhibits increased aggressiveness with poor prognosis in men diagnosed with the disease. These incongruent observations led us to explore the role of androgen and androgen receptor in this disease. We found an approximately 70% decrease in median AR expression (p<0.0001) in 24 PTC patient tissue samples, compared to matched, normal thyroid tissue. Preliminary data from our lab indicate that androgen receptor (AR) acts as a negative regulator of growth as evidenced by a statistically significant 48% decrease in proliferation over 72 hours upon 5α-dihydrotestosterone (DHT) addition to 8505c anaplastic/PTC cells stably transfected with AR (clone 84E7). Transcriptional profiling using RNAseq and gene ontology analysis, on 48 hour DHT treated 84E7 cells revealed significant changes in gene expression associated with proliferation (474 genes, p = 2.4E-24) and cell cycle progression (129 genes, p = 6.54E-6). Continuous AR activation (3 to 6 days) resulted in G1 growth arrest, not accompanied by cell death, but rather a flattened, vacuolized cell morphology, indicative of senescence. This was substantiated by an increase in SA-βGal positivity from background 2.47% to approximately 65.5%, which was attenuated by the AR antagonist, flutamide. Three to six day DHT exposure resulted in increased total RNA and protein content measured using Acridine Orange and Sulforhodamine B, respectively. Senescent 84E7 cells failed to resume growth when transferred into DHT-free medium for 3 days, suggesting a permanent growth arrest. Reactive oxygen species (ROS), implicated in Rb-mediated senescence, were found to be doubled by day 6 of AR activation, compared to control cells. Additionally, using flow cytometry, we found increased p21, p27, cyclin D1, FOXO1, and phosphorylation of FOXO1 by days 3-6, suggesting that AR-dependent senescence is mediated via p21 and p27 by FOXO1/3 proteins. Furthermore, cytokine profiling of the senescence-associated secretory phenotype (SASP) would aid in defining the pro-tumorigenic or tumor-suppressive potential of androgen-induced senescent thyroid cancer cells. Our study elucidates the induction of senescence as a novel function of AR activation in thyrocytes and may indicate a protective role of AR activation in the decreased incidence of thyroid cancer in men. Citation Format: Anvita Gupta, Melanie Jones, Timmy O’Connell, Dorota Halicka, Jiangwei Li, Hong Zhao, Augustine Moscatello, Zbigniew Darzynkiewicz, Raj Tiwari, Jan Geliebter. Continuous androgen receptor stimulation in thyroid cancer cells induces irreversible senescence. [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 2841.

Abstract 4576: Hepatocyte nuclear factor 4 alpha suppresses cell proliferation and induces cellular senescence and apoptosis in prostate cancer cells

Abstract Hepatocyte nuclear factor 4α (HNF4α, NR2A1) is a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors. Recent advances show that it is a key transcriptional regulator of many genes, involved in xenobiotic and drug metabolism and also cancers of gastrointestinal tract. However, the exact functional roles of HNF4α gene in prostate cancer progression are still not fully understood. In this study we used the overexpression (gain-of-function) and knockdown (loss-of-function) approaches to determine the functional roles of HNF4α in prostate cancer. Our results showed that HNF4α exhibited a reduced expression pattern in many prostate cancer cells compared to immortalized prostatic cell lines. Its overexpression could significantly inhibit the cell proliferation of prostate cancer cells, and trigger the cellular senescence and apoptosis by activation of p21 signal pathway in a p53-independent manner. We showed that stable overexpression of HNF4α could significantly induce G2/M arrest in PC3 cells, while shRNA-mediated knockdown of HNF4α could induce G1 growth arrest, suggesting that HNF4α might play a negative role in the cell cycle regulation of PC3 cells. Moreover, stable HNF4α knockdown could confer resistance to paclitaxel treatment and enhance colony formation capacity in prostate cancer cells. Together, our results suggest that HNF4α may play a tumor suppressor function in prostate cancer progression.This work was supported by a Direct Grant for Research 2015-2016 (2015. 1. 064), CUHK. Citation Format: Zhu WANG, Franky Leung Chan. Hepatocyte nuclear factor 4 alpha suppresses cell proliferation and induces cellular senescence and apoptosis in prostate cancer cells. [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 4576.

MP83-02 ADDITION OF EMETINE DIHYDROCHLORIDE TO PLATINUM BASED CHEMOTHERAPY INHIBITS BLADDER CANCER CELL PROLIFERATION AND REDUCES THE EFFECTIVE DOSE OF PLATINUM CHEMOTHERAPY

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP83-02 ADDITION OF EMETINE DIHYDROCHLORIDE TO PLATINUM BASED CHEMOTHERAPY INHIBITS BLADDER CANCER CELL PROLIFERATION AND REDUCES THE EFFECTIVE DOSE OF PLATINUM CHEMOTHERAPY Valerie J. Davidson, Robert H. Blackwell, Kimberly E. Foreman, Paul C. Kuo, Robert C. Flanigan, and Gopal N. Gupta Valerie J. DavidsonValerie J. Davidson More articles by this author , Robert H. BlackwellRobert H. Blackwell More articles by this author , Kimberly E. ForemanKimberly E. Foreman More articles by this author , Paul C. KuoPaul C. Kuo More articles by this author , Robert C. FlaniganRobert C. Flanigan More articles by this author , and Gopal N. GuptaGopal N. Gupta More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2184AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Emetine is a natural alkaloid that inhibits protein synthesis at the micromolar level. Decades ago, Phase I and II clinical trials of emetine as a single agent showed modest efficacy against many solid tumors, but these findings were not pursued. We have previously reported that low, nanomolar concentrations of emetine act synergistically with cisplatin to inhibit invasive bladder cancer cell proliferation. Here, we extend these findings to evaluate the addition of emetine to the standard of care chemotherapeutic regimen of cisplatin plus gemcitabine. Because 50% of patients are unfit for cisplatin-based therapies, we also examined the effect of emetine combined with carboplatin and gemcitabine on bladder cancer cell proliferation. METHODS Normal urothelial cells and UMUC3, HT1376, and T24 invasive bladder cancer cell lines were cultured under standard conditions. Cells were treated with gemcitabine, cisplatin, carboplatin, and emetine alone or in combination at concentrations based on the drug's experimentally determined inhibitory concentration-50 (IC50). Proliferation was measured via MTT assay or trypan blue-staining based cell counts. Cell cycle data was obtained via DNA-propidium iodide staining and flow cytometry. RESULTS The addition of emetine to either cisplatin-gemcitabine or carboplatin-gemcitabine significantly decreased cell proliferation in cancer cells. Normal urothelial cells were more resistant with ten-fold higher concentrations of emetine required to observe the same effect on proliferation. Cell cycle analysis revealed the addition of emetine did not cause cell death, but rather induced a G1 growth arrest. This growth arrest was irreversible at cisplatin or carboplatin concentrations at or above the calculated IC50. CONCLUSIONS Addition of emetine to either cisplatin or carboplatin-based chemotherapeutic regimens resulted in a significant decrease in bladder cancer cell proliferation. Our findings suggest the addition of emetine to the current standard of care therapies may benefit patients. Importantly, inclusion of emetine appears to lower the dose of cisplatin or carboplatin required to block cell proliferation. This may allow some patients previously unfit for cisplatin-gemcitabine to qualify for this therapeutic regimen. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1081 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Valerie J. Davidson More articles by this author Robert H. Blackwell More articles by this author Kimberly E. Foreman More articles by this author Paul C. Kuo More articles by this author Robert C. Flanigan More articles by this author Gopal N. Gupta More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism.

Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies.

Induction of miR-137 by Isorhapontigenin (ISO) Directly Targets Sp1 Protein Translation and Mediates Its Anticancer Activity Both In Vitro and In Vivo.

Our recent studies found that isorhapontigenin (ISO) showed a significant inhibitory effect on human bladder cancer cell growth, accompanied with cell-cycle G0-G1 arrest as well as downregulation of Cyclin D1 expression at transcriptional level via inhibition of Sp1 transactivation in bladder cancer cells. In the current study, the potential ISO inhibition of bladder tumor formation has been explored in a xenograft nude mouse model, and the molecular mechanisms underlying ISO inhibition of Sp1 expression and anticancer activities have been elucidated both in vitro and in vivo. Moreover, the studies demonstrated that ISO treatment induced the expression of miR-137, which in turn suppressed Sp1 protein translation by directly targeting Sp1 mRNA 3'-untranslated region (UTR). Similar to ISO treatment, ectopic expression of miR-137 alone led to G0-G1 cell growth arrest and inhibition of anchorage-independent growth in human bladder cancer cells, which could be completely reversed by overexpression of GFP-Sp1. The inhibition of miR-137 expression attenuated ISO-induced inhibition of Sp1/Cyclin D1 expression, induction of G0-G1 cell growth arrest, and suppression of cell anchorage-independent growth. Taken together, our studies have demonstrated that miR-137 induction by ISO targets Sp1 mRNA 3'-UTR and inhibits Sp1 protein translation, which consequently results in reduction of Cyclin D1 expression, induction of G0-G1 growth arrest, and inhibition of anchorage-independent growth in vitro and in vivo. Our results have provided novel insights into understanding the anticancer activity of ISO in the therapy of human bladder cancer.