Concentration-dependent Decrease In Proliferation Research Articles

Heavy Water Affects Vital Parameters of Human Melanoma Cells in vitro.

PurposeAlthough regular water is composed of two hydrogens and one oxygen, referred to as H2O, a small amount of water on this planet contains alternative forms of elements with different molecular weights because of the addition of neutrons. The present study was dedicated to studying the effect of heavy water (D2O), in which the two hydrogens become substituted by deuterium, on the cell physiology of different human cells with particular focus on malignant melanoma cells.MethodsCells were cultured in regular medium in which the content of H2O was gradually substituted by D2O or deuterium-depleted water (DDW). Following this, the changes of basic cellular parameters, such as morphology, migration, proliferation, cell cycle, apoptosis and microtubule integrity were examined.ResultsIt was found that raising the D2O content above the standard levels led to a concentration-dependent decrease in proliferation. Lowering the D2O levels below this level had no effect. Likewise, elevated D2O levels hampered migration. Moreover, cell-cycle analysis showed an increase of sub-G1 cells. Corroboratively, markers for apoptosis were induced (histone-associated DNA fragments, Bax, and PARP). In regard to microtubule integrity, only very high levels of D2O (75%) caused partial filament condensation.ConclusionD2O, although chemically identical with H2O, shows proapoptotic and antiproliferative effects on melanoma cells. These findings give a closer look of this interesting compound.

Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

BackgroundEndocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility.MethodsA panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1wt or ESR1Y537S, modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies.ResultsHere, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001.ConclusionsOur data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER.

Abstract P4-04-09: New oral SERD elacestrant (RAD1901) shows efficacy in breast cancer models harbouring ESR1 mutations and enhances the antiproliferative activity of mTORC1 and CDK4/6 inhibitors

Abstract Background: Targeting estrogen receptor (ER) signalling is the main therapeutic option for ER+ breast cancer (BC). However, over 30% of patients relapse with endocrine resistance emphasising the need for improved therapeutic strategies. The prevalence of ESR1 mutations in relapsed tumours highlights the sustained reliance on ER signalling, rationalising continued targeting of ER. Unlike other endocrine therapies such as aromatase inhibitors (AI) and tamoxifen, selective ER degraders (SERDs) are competitive ER antagonists, that induce a conformational change in ER resulting in ubiquitination and degradation via the proteasomal pathway. Fulvestrant has shown clinical utility in advanced BC but is limited by its pharmaceutical properties highlighting the need for SERDs with enhanced bioavailability and pharmacokinetic properties. Here, we show that elacestrant (RAD1901) an orally bioavailable SERD, has antitumor activity in endocrine sensitive and resistant models of ER+ BC. Furthermore, elacestrant enhances the efficacy of mTORC1 inhibitor, everolimus and CDK4/6 inhibition, in model systems. Methods: Several ER+ BC lines adapted to long-term E deprivation (LTED) and harbouring wild-type or a naturally occurring ESR1 mutation, were treated with elacestrant or fulvestrant +/- estradiol (E2). Effects on cell proliferation, cell signalling, cell cycle, transcription, ER protein stability and ER genomic binding were assessed. Efficacy in combination with everolimus, palbociclib and abemaciclib was also evaluated. Results: Cell proliferation assays in 2D and spheroids in the presence of 0.01nM E2 showed a concentration-dependent decrease in proliferation in response to elacestrant and fulvestrant. GI50 values for elacestrant in general were 10-fold higher than fulvestrant but equated to doses that are clinically achievable for the drug. Most importantly, elacestrant suppressed proliferation of two LTED models harbouring ESR1 mutations, MCF7 LTEDY537C (GI50 5nM) and SUM44-LTEDY537S (GI50 100nM). GI50 values of elacestrant and fulvestrant showed similar reduction of ER, progesterone receptor (PGR) and cyclinD1 together with decreased phosphorylation of retinoblastoma (RB), concordant with cell cycle arrest. Chromatin immunoprecipitation (ChIP) for ER in response to elacestrant or fulvestrant showed a reduction in recruitment of ER to TFF1, GREB1 and PGR promoters and concomitant reduction in mRNA expression of these genes in the presence of E2. Elacestrant-mediated ER depletion was dependent on the 26S proteasome, as addition of the proteasome inhibitor MG132, fully blocked elacestrant depletion of ER, similar to the effect of MG132 treatment in preventing fulvestrant-mediated ER turnover. The combination of elacestrant with CDK4/6 inhibitors, palbociclib or abemaciclib, demonstrated additivity compared with monotherapy. In addition, elacestrant inhibited growth of palbociclib-resistant MCF7 LTED cells. Conclusion: These findings highlight the potential utility of elacestrant as a 1st or 2nd line drug in the treatment of ER+ BC. The results support the testing of elacestrant versus fulvestrant after relapse on an AI, either alone or in combination with a CDK4/6 inhibitor. Citation Format: Martin L-A, Pancholi S, Simigdala N, Nikitorowicz-Buniak J, Ribas R, Garner F, Bihani T, Dowsett M. New oral SERD elacestrant (RAD1901) shows efficacy in breast cancer models harbouring ESR1 mutations and enhances the antiproliferative activity of mTORC1 and CDK4/6 inhibitors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-04-09.

Abstract 4157: Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity

Abstract Introduction The majority of breast cancers (BC) are estrogen (E) receptor positive (ER+). Endocrine therapies target E stimulation of tumour growth but resistance remains problematic, often a result of enhanced crosstalk between ER and growth factor pathways. Previously we reported the antiproliferative efficacy of combining everolimus (RAD001, mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB and mTORC1) may target tumour rewiring and provide enhanced long-term clinical utility in endocrine resistant BCs. Methods Several ER+ BC lines adapted to long term E deprivation (LTED), modelling relapse to an aromatase inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan ERBB inhibitor) in the presence or absence of estradiol (E2), tamoxifen or fulvestrant. Effects on proliferation, cell signalling, cell cycle and transcription were assessed. Additionally, an in vivo model of AI resistance was treated with monotherapies or combinations to evaluate efficacy in delaying tumour progression. Results All cell lines showed dose dependent decreases in proliferation in response to RAD001 (IC50 0.6-50nM without E2; 1-10nM with E2). Neratinib showed a wide range of IC50 values in the presence of E2 (300-1000nM). In the absence of E2, wild type (wt) cell lines showed IC50 values in excess of 1800nM with hormetic response curves, whilst in the LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in both wt and LTED lines but the maximum effect was observed with a triple combination of RAD001, neratinib and endocrine therapy. Expression of pS6 was suppressed by RAD001 in all cells tested, whilst neratinib caused a cell specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines upon treatment with RAD001. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and reduced cell cycle progression. In the absence of E2, RAD001 reduced ER mediated transcription and recruitment of ER and CREB binding protein to the TFF1 promoter, contrasting with neratinib, which caused a marked increase. In vivo study using LTED tumour xenografts showed the triple combination of RAD001, neratinib and fulvestrant was the most effective at reducing tumour volume (89%, p<0.001). Antitumor effects persisted after therapy removal with exception of RAD001 alone or in combination with neratinib where a slow increase in tumour growth was observed. Conclusion The combination of RAD001, neratinib and endocrine agents may be effective in patients who have relapsed on endocrine therapy but retain a functional ER by combating ERBB pathway upregulation. Citation Format: Ricardo Ribas, Sunil Pancholi, Stephanie K. Guest, Aradhana Rani, Joanna Nikitorowicz-Buniak, Nikiana Simigdala, Allan Thornhill, Richard E. Cutler Jr, Alshad S. Lalani, Francesca Avogadri-Connors, Mitch Dowsett, Stephen R. Johnston, Lesley-Ann Martin. Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity [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 4157. doi:10.1158/1538-7445.AM2017-4157

Abstract 4160: Impact of oral selective estrogen receptor degrader RAD1901 in preclinical models of endocrine sensitive/resistant breast cancer

Abstract Aromatase inhibitors (AI) are the mainstay for treatment of postmenopausal estrogen receptor positive (ER+) primary breast cancer (BC). However, many patients relapse whilst retaining a functional ER. The selective ER degrader (SERD) fulvestrant has been evaluated as a potential 2nd or 3rd line therapy for patients who relapse on AI treatment. Although exhibiting promising potential, its low bioavailability has limited its use to combination therapy in metastatic patients. Here, we assessed the efficacy of the orally available SERD, RAD1901, in a panel of BC cells with varying genetic backgrounds modelling patients both sensitive and resistant to AI therapy (LTED). Fulvestrant was used in parallel to allow relative responses to be compared. Cell proliferation assays in 2D and spheroids the presence of 0.01nM 17β-estradiol showed a concentration-dependent decrease in proliferation in response to RAD1901 and fulvestrant. GI50 values for RAD1901 in general were 10-fold higher than fulvestrant but equated to doses that are clinically achievable for RAD1901. Most importantly, RAD1901 effectively suppressed proliferation of two LTED models harbouring naturally occurring ESR1 mutations, MCF7 LTEDY537C (GI50 5nM) and SUM44-LTEDY537S (GI50 100nM). GI50 values of RAD1901 and fulvestrant showed similar reduction of ER, progesterone receptor (PGR) and cyclin D1 together with decreased phosphorylation of retinoblastoma (RB), concordant with cell cycle arrest. Furthermore, chromatin immunoprecipitation (ChIP) for ER in response to RAD1901 or fulvestrant showed a 70% reduction in recruitment of ER to TFF1, GREB1 and PGR promoters and concomitant reduction in mRNA expression of these genes. In MCF7-Arom cells, combination of letrozole with RAD1901 or fulvestrant showed enhanced antiproliferative effect compared to letrozole alone. The addition of RAD1901 to CDK4/6 inhibitor palbociclib or abemaciclib demonstrated additivity compared with monotherapy. In addition, RAD1901 effectively inhibited growth of palbociclib-resistant MCF7 LTED cells.These preclinical findings highlight the potential utility of RAD1901 as a potent drug in the treatment of ER+ BC. Combined with its bioavailability profile, RAD1901 warrants clinical testing versus fulvestrant after relapse on an AI, either alone or in combination with a CDK4/6 inhibitor. Citation Format: Sunil Pancholi, Joanna Nikitorowicz-Buniak, Ricardo Ribas, Nikiana Simigdala, Fiona Garner, Teeru Bihani, Stephen R. Johnston, Mitch Dowsett, Lesley-Ann Martin. Impact of oral selective estrogen receptor degrader RAD1901 in preclinical models of endocrine sensitive/resistant breast cancer [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 4160. doi:10.1158/1538-7445.AM2017-4160

495. Activation of DNA Pattern Recognition Receptors After Plasmid Electrotransfer in Melanoma Cells and Tumors

In vivo electroporation or electrotransfer, the application of controlled electric pulses, enhances delivery of plasmid DNA to a wide variety of healthy tissues as well as many tumor types. Electrotransfer of pDNA encoding therapeutic genes substantially increases gene expression, enhancing subsequent therapeutic effects. Delivery of therapeutic plasmid DNA has reached clinical trials in the US and in Europe, primarily for cancer therapies and infectious disease vaccines. In several preclinical tumor models, delayed tumor growth, increased survival time, and even complete tumor regression can occur with intratumoral electroporation, also known as electrotransfer, of DNA oligonucleotides or plasmid DNA devoid of a therapeutic gene (empty vector). In B16.F10 mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines and chemokines including IFNβ, implicating the binding and activation of intracellular DNA-specific pattern recognition receptors in response to DNA electrotransfer. The purpose of this study was to investigate whether melanoma tumors and cells express cytosolic DNA sensors and whether these sensors respond to pDNA electrotransfer. Histologically, tumor necrosis independent of caspase-3 was observed. Although the mRNAs for several DNA sensors were detected in tumors, none was significantly upregulated. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated after pDNA electrotransfer. The mRNAs for several DNA sensors were present in these cells and DAI, DDX60, and p204 mRNAs were significantly upregulated after pDNA electrotransfer. DDX60 protein levels were coordinately upregulated. Mirroring the observation of tumor necrosis, cells underwent a significant pDNA concentration-dependent decrease in proliferation and survival. Taken together, increased IFNβ and DNA sensor expression accompanied by cell death and tumor necrosis indicate that pDNA electrotransfer activates intracellular DNA sensors in B16.F10 cells and tumors, producing both in vitro and in vivo effects. The absence of activation of DNA sensors in vivo could be due to the lower transfection efficiency compared to that in vitro or to dilution by other tumor cell types. Electrotransfer is an efficient means of enhancing plasmid DNA introduction into tissues such as skin, muscle, and tumors for therapeutic application. Localized inflammation and induced cell death may contribute to cancer gene therapies but may impede gene therapies for which these effects are not desirable.

Plasmid electrotransfer activates DNA pattern recognition receptors and induces production of proinflammatory molecules

Abstract Electroporation or electrotransfer is an effective delivery system for introducing plasmid DNA into cells and tissues. During this process, pDNA primarily enters the cell via endocytosis, although some DNA reaches the cytosol. Endosomal, cytosolic, and nuclear DNA-specific pattern recognition receptors (PRRs) in many cell types have been described. Activated PRRs induce the production of proinflammatory molecules and programmed cell death. In B16.F10 mouse melanomas, electrotransfer of empty backbone plasmid induced the production of several proinflammatory cytokines and chemokines including IFNβ, implicating the binding and activation of intracellular DNA-specific PRRs. Histologically, tumor necrosis independent of caspase-3 was observed. The purpose of this study was to investigate whether tumors and cells express cytosolic DNA sensors and whether these sensors respond to pDNA electrotransfer. Although the mRNAs for several DNA sensors were detected in tumors, none was significantly upregulated. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated after DNA electrotransfer. The mRNAs for several DNA sensors were present in these cells and DAI, DDX60, and p204 mRNAs were significantly upregulated after pDNA electrotransfer. DDX60 protein levels were coordinately upregulated. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, increased IFNβ and DNA sensor expression accompanied by cell death and tumor necrosis indicate that DNA electrotransfer activates intracellular DNA sensors in B16.F10 cells and tumors, producing both in vitro and in vivo effects.

Abstract P3-05-05: Targeting tumor re-wiring by triple blockade of mTORC1, ERBB and ER signaling pathways in endocrine resistant breast cancer

Abstract AIM To target tumor re-wiring by combined mTORC1 inhibition plus hormonal treatment with or without co-blockade of ERBB signaling in endocrine resistant models of human breast cancer (BC). BACKGROUND Around 80% of BCs are estrogen receptor positive (ER+). Endocrine therapies target estrogenic stimulation of tumor growth but resistance remains problematic. Several strategies have shown that resistance often depends on the acquisition of enhanced cross-talk between ER and growth-factor pathways, allowing the disease to circumvent the need for steroid hormones. We have previously reported the antiproliferative effects of the combination of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signaling were observed. We hypothesised that combined targeting of three signaling pathways, namely ER, ERBB and mTORC1 may provide enhanced anti-tumor activity. METHODS ER+ BC cell lines (MCF7, SUM44 and HCC1428) adapted to long term estrogen-deprivation (LTED) which model relapse on an aromatase inhibitor, along with their wild-type (wt) cell lines were treated with neratinib, a pan-ERBB tyrosine kinase inhibitor, in combination with RAD001 ± estradiol (E2), tamoxifen or fulvestrant. End points included proliferation, cell signaling, cell cycle and effect on ER-mediated transactivation and recruitment by ChIP. RESULTS All cell lines showed a concentration-dependent decrease in proliferation in response to RAD001 (IC50 0.6-50nM in absence of E2 and 1-10nM in presence of E2). A wide range of IC50 values (300-1000nM) was observed with neratinib treatment in the presence of E2. However, in the absence of E2, wt cell lines showed IC50 values in excess of 1800nM with hormetic dose response curves, in which lower concentrations induced cell proliferation. In contrast, LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in all wt cell lines and their LTED derivatives, but the maximum effect was observed when a triple combination of RAD001, neratinib and ER-blockade was used. Expression of pS6 was dramatically suppressed by RAD001 ± neratinib in all cell lines tested, whilst neratinib caused a cell line specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines following treatment with RAD001, indicating both inhibitors were effectively suppressing their respective targets. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and significantly reduced cell cycle progression. In the absence of E2, RAD001 caused a reduction in ER-mediated transcription and decrease in recruitment of ER and the CREB-binding protein (CBP) to the TFF1 promoter. In contrast, neratinib induced a marked increase in ER-recruitment and concomitant rise in ER-mediated transactivation, which was reduced by the addition of RAD001. CONCLUSION Targeting tumor re-wiring by triple blockade of ERBB, ER and mTORC1 signaling pathways significantly reduces cell proliferation supporting the potential combination in patients who have relapsed on endocrine therapy and retain a functional ER. Citation Format: Nikitorowicz-Buniak J, Ribas R, Rani A, Pancholi S, Guest SK, Cutler Jr RE, Lalani A, Dowsett M, Johnston SR, Martin L-A. Targeting tumor re-wiring by triple blockade of mTORC1, ERBB and ER signaling pathways in endocrine resistant 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 P3-05-05.

Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells.

In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.

Abstract 2750: Conditional RNAi-mediated knockdown of TPX2 synergizes with vincristine and docetaxel cytotoxicity against HeLa cell sub-clones in vitro and in vivo

Abstract Microtubuli-associated proteins could be potent novel targets for anti-cancer therapy. To test this hypothesis, a HeLa cell line was generated, in which the reverse tetracycline-dependent transactivator rtTA2S-M2 drives the expression of eGFP and a miRNA, inducing RNAi-dependent inhibition of TPX2 expression, from the bidirectional tet-regulated promoter (HeLa EM2-11-TPX2). As a negative control for respective experiments a similar cell line was generated (HeLa EM2-11), in which the presence of the inducer doxycycline (dox) activates expression of the genes mCherry and firefly luciferase. First, the effect of TPX2 knockdown on in vitro proliferation was determined by MTT assay. Here, the respective cells were treated with either a single agent therapy consisting of vincristine (VC) (0.5-2.6 ng/ml) or docetaxel (DCX) (0.125-4 nmol/l) or dox (0.2 μg/ml), or a combination of dox and VC or DCX, and cell viability was assessed after 72h. Treatment with VC or DCX caused a concentration-dependent decrease in proliferation in vitro, whereas knockdown of TPX2 reduced cell growth by 35-45%. The proliferation of HeLa EM2-11-TPX2 was synergistically reduced by the combination treatment dox and VC and additively reduced by dox and DCX when compared to the single agent exposures. Next, the efficiency of tumor growth inhibition following knockdown of TPX2 alone or in combination with VC or DCX was tested in a nude mouse xenograft model. Here, the size of tumors growing in nude mice following s.c. implantation was monitored over a period of 4-5 weeks. Dox (0.2 mg/ml in drinking water) was given continuously from day 14 after cell injection until the end of the observation period. The antimitotic agents VC (1 mg/kg) or DCX (22.3 mg/kg) were applied twice on days 14 and 21. In nude mice, TPX2-knockdown alone caused a 71% reduction in tumor growth. VC or DCX reduced tumor growth by 59% or 76%, respectively. The combination of dox and VC or DCX inhibited tumor growth by 93% or 97%, respectively. Both combination treatments were significantly superior to the respective single treatment arms (p<0.05). We could demonstrate that persistent inactivation of TPX2 expression was highly effective in inhibiting tumor growth, both in vitro and in vivo. The combination of the conditional TPX2-knockdown and VC showed a synergistic therapeutic effect in vitro, whereas the combination of TPX2-knockdown and DCX showed an additive therapeutic effect in vitro. Both combination treatments were significantly superior to the respective single agent treatments in vivo. It is concluded that TPX2 is a valuable treatment target, causing effective tumor growth inhibition, which can be successfully combined with other anti-mitotic agents. Citation Format: Annette J. Allmendinger, Kai Schönig, Oliver J. Gruss, Stefan Berger, Martin R. Berger. Conditional RNAi-mediated knockdown of TPX2 synergizes with vincristine and docetaxel cytotoxicity against HeLa cell sub-clones in vitro and in vivo. [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 2750. doi:10.1158/1538-7445.AM2014-2750

MycobacteriumBovisOrnithine Carbamoyltransferase, MB1684, Induces Proinflammatory Cytokine Gene Expression by Activating NF-κB in Macrophages

Mycobacterium bovis is the etiological factor of bovine tuberculosis (BTB), posing a significant problem to domestic cattle. The bacterium is also zoonotic, affecting human health worldwide. Macrophage evasion of the bacterium involves mycobacterial molecules such as MB1684 (ornithine carbamoyltransferase). In this study, we confirmed a concentration-dependent decrease in proliferation of Ana-1 macrophages when treated with rMB1684 when compared with mycobacterium bovis purified protein derivative of tuberculosis (MbPPD) or phosphate buffer solution incubation groups. We examined the activation of nuclear factor-kappa B (NF-κB) upon exposure to MB1684, and its role in MB1684-induced upregulation of interferon (IFN)-γ and proinflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor-α) in Ana-1 macrophages. The levels of proinflammatory cytokines and IFN-γ were significantly high in MB1684-treated Ana-1 macrophages. The treatment led to an increase in NF-κB activation and a high expression of the just mentioned proinflammatory cytokines. NF-κB inhibition significantly abrogated MB1684-induced upregulation of proinflammatory cytokine mRNA expression, which suggests that MB1684-induced activation of NF-κB in turn stimulates gene expression of IFN-γ and proinflammatory cytokines in Ana-1 macrophages. The experiment was repeated in bone marrow macrophages, a more in-vivo-like model system, and similar results validated our conclusion. Further, we identified the possibility of the application of MB1684 antigen for the detection of BTB in cattle serum.

Effectiveness and molecular interactions of the clinically active mTORC1 inhibitor everolimus in combination with tamoxifen or letrozole in vitro and in vivo

IntroductionStrategies to improve the efficacy of endocrine agents in breast cancer (BC) therapy and to delay the onset of resistance include concomitant targeting of the estrogen receptor alpha (ER) and the mammalian target of rapamycin complex 1 (mTORC1), which regulate cell-cycle progression and are supported by recent clinical results.MethodsBC cell lines expressing aromatase (AROM) and modeling endocrine-sensitive (MCF7-AROM1) and human epidermal growth factor receptor 2 (HER2)-dependent de novo resistant disease (BT474-AROM3) and long-term estrogen-deprived (LTED) MCF7 cells that had acquired resistance associated with HER2 overexpression were treated in vitro and as subcutaneous xenografts with everolimus (RAD001-mTORC1 inhibitor), in combination with tamoxifen or letrozole. End points included proliferation, cell-cycle arrest, cell signaling, and effects on ER-mediated transactivation.ResultsEverolimus caused a concentration-dependent decrease in proliferation in all cell lines, which was associated with reductions in S6 phosphorylation. Everolimus plus letrozole or tamoxifen enhanced the antiproliferative effect and G1-accumulation compared with monotherapy, as well as increased phosphorylation (Ser10) and nuclear accumulation of p27 and pronounced dephosphorylation of Rb. Sensitivity was greatest to everolimus in the LTED cells but was reduced by added estrogen. Increased pAKT occurred in all circumstances with everolimus and, in the BT474 and LTED cells, was associated with increased pHER3. Decreased ER transactivation suggested that the effectiveness of everolimus might be partly related to interrupting cross-talk between growth-factor signaling and ER. In MCF7-AROM1 xenografts, letrozole plus everolimus showed a trend toward enhanced tumor regression, versus the single agents. In BT474-AROM3 xenografts, everolimus alone was equally effective at reducing tumor volume as were the combination therapies.ConclusionsThe results provide mechanistic support for recent positive clinical data on the combination of everolimus and endocrine therapy, as well as data on potential routes of escape via enhanced HER2/3 signaling. This merits investigation for further improvements in treatment efficacy.

A High Omega-3 Fatty Acid Diet Mitigates Murine Pancreatic Precancer Development

Diets containing omega-3 (ω-3) fat have been associated with decreased tumor development in the colon, breast, and prostate. We assessed the effects of a diet rich in ω-3 fat on the development of pancreatic precancer in elastase (EL)-Kras transgenic mice and examined the effect of an ω-3 fatty acid on pancreatic cancer cells in vitro. Two cohorts of EL-Kras mice were fed a high ω-3 fat diet (23% menhaden oil) for 8 and 11 mo and compared with age-matched EL-Kras mice fed standard chow (5% fat). Pancreata from all mice were scored for incidence and frequency of precancerous lesions. Immunohistochemistry was performed for proliferating cell nuclear antigen (PCNA) to assess proliferative index in lesions of mice fed either a high ω-3 or standard diet. In vitro, the effect of the ω-3 fatty acid, docosahexaenoic acid (DHA), on two pancreatic cancer cell lines was assessed. Cancer cell proliferation was assessed with an MTT assay; cell cycle analysis was performed by flow cytometry; and apoptosis was assessed with annexin/PI staining. The incidence, frequency, and proliferative index of pancreatic precancer in EL-Kras mice was reduced in mice fed a high ω-3 fat diet compared with mice fed a standard chow. In vitro, DHA treatment resulted in a concentration-dependent decrease in proliferation through both G1/G0 cell cycle arrest and induction of apoptosis. A high ω-3 fat diet mitigates pancreatic precancer by inhibition of cellular proliferation through induction of cell cycle arrest and apoptosis.

3,3'-Diindolylmethane inhibits angiogenesis and the growth of transplantable human breast carcinoma in athymic mice

Studies have linked the consumption of broccoli and other cruciferous vegetables to a reduced risk of breast cancer. The phytochemical indole-3-carbinol (I3C), present in cruciferous vegetables, and its major acid-catalyzed reaction product 3,3'-diindolylmethane (DIM) have bioactivities relevant to the inhibition of carcinogenesis. In this study, the effect of DIM on angiogenesis and tumorigenesis in a rodent model was investigated. We found that DIM produced a concentration-dependent decrease in proliferation, migration, invasion and capillary tube formation of cultured human umbilical vein endothelial cells (HUVECs). Consistent with its antiproliferative effect, which was significant at only 5 microM DIM, this indole caused a G1 cell cycle arrest in actively proliferating HUVECs. Furthermore, DIM downregulated the expression of cyclin-dependent kinases 2 and 6 (CDK2, CDK6), and upregulated the expression of CDK inhibitor, p27(Kip1), in HUVECs. We observed further in a complementary in vivo Matrigel plug angiogenesis assay that, compared with vehicle control, neovascularization was inhibited up to 76% following the administration of 5 mg/kg DIM to female C57BL/6 mice. Finally, this dose of DIM also inhibited the growth of human MCF-7 cell tumor xenografts by up to 64% in female athymic (nu/nu) mice, compared with the vehicle control. This is the first study to show that DIM can strongly inhibit the development of human breast tumor in a xenograft model and to provide evidence for the antiangiogenic properties of this dietary indole.