MCF-7 Xenograft Model Research Articles

RAD1901: a novel, orally bioavailable selective estrogen receptor degrader that demonstrates antitumor activity in breast cancer xenograft models.

Agents that inhibit estrogen production, such as aromatase inhibitors or those that directly block estrogen receptor (ER) activity, such as selective estrogen receptor modulators and selective estrogen receptor degraders, are routinely used in the treatment of ER-positive breast cancers. However, although initial treatment with these agents is often successful, many women eventually relapse with drug-resistant breast cancers. To overcome some of the challenges associated with current endocrine therapies and to combat the development of resistance, there is a need for more durable and more effective ER-targeted therapies. Here we describe and characterize a novel, orally bioavailable small-molecule selective estrogen receptor degrader, RAD1901, and evaluate its therapeutic potential for the treatment of breast cancer. RAD1901 selectively binds to and degrades the ER and is a potent antagonist of ER-positive breast cancer cell proliferation. Importantly, RAD1901 produced a robust and profound inhibition of tumor growth in MCF-7 xenograft models. In an intracranial MCF-7 model, RAD1901-treated animals survived longer than those treated with either control or fulvestrant, suggesting the potential benefit of RAD1901 in the treatment of ER-positive breast cancer that has metastasized to the brain. Finally, RAD1901 preserved ovariectomy-induced bone loss and prevented the uterotropic effects of E2, suggesting that it may act selectively as an agonist in bone but as an antagonist in breast and uterine tissues. RAD1901 is currently under clinical study in postmenopausal women with ER-positive advanced breast cancer.

Abstract 4719: Targeting interdependent signaling pathways to increase the durability and magnitude of response: promising combination therapy with dual mTORC1/2 inhibitors and CDK4/6 inhibitors

Abstract Two emerging mechanisms of endocrine resistance in estrogen receptor positive (ER+) breast cancers include the activation of the phosphatidylinositol 3-kinase (PI3K) / mammalian target of rapamycin (mTOR) pathway and the de-coupling of cell cycle control from ER signaling via de-regulation of the cyclin D/cyclin dependent kinase (CDK4/6) pathway. In this study, we hypothesized that combining inhibitors of both pathways using the dual mTORC1 and mTORC2 inhibitor AZD2014 and the CDK4/6 inhibitor palbociclib would elicit an improved tumor response over agents that inhibit either pathway alone. Moreover, we hypothesized that combined inhibition of CDK4/6 and TORC1/2 together with inhibition of ER signaling, would cause a profound anti-tumor effect in breast cancer models. In breast cancer cell lines, the combination of AZD2014 and palbociclib caused a synergistic inhibitory effect on cell growth. These effects occurred under conditions where addition of 300nM AZD2014 resulted in significant blockade on both TORC1 and TORC2 downstream effectors (>80% inhibition of p-AKT, p-S6 and p-4EBP1) as well as significant down-regulation of cyclin D1 levels (>70%). Similarly, inhibition of CDK4/6 using palbociclib (300nM) caused >80% inhibition of p-RB and subsequent cell cycle blockade.The effects observed in breast cancer cell lines were recapitulated in vivo using the MCF7 xenograft model, where tumor regressions (>105%) were observed with the combination. Furthermore, combining AZD2014, palbociclib and fulvestrant in this model also caused tumor regressions.To assess bone marrow tolerability of this combination, we investigated the response of human bone marrow multipotent progenitors (CD34+) in vitro. Palbociclib caused transient cell cycle G1 arrest. The combination of palbociclib with AZD2014 delayed entry into cell cycle, but did not have significant impact on the cell viability (<15%). We hypothesized that inhibition of CDK4/6 in combination with AZD2014 would also significantly affect pathway outputs and the transcriptional events downstream of the transcription factors E2F (e.g. CENPE, CCNA2, CDC6 and E2F1) and ER (e.g. PR). We therefore measured specific gene signatures downstream of these receptors in breast cancer cell lines. The ability to dose AZD2014 intermittently compared with rapalogues, together with its ability to block signaling from both TORC1 and 2, make this compound an ideal candidate for combining with CDK4/6 inhibitors such as palbociclib. Furthermore, addition of anti-hormonal therapies such as fulvestrant to this combination may provide additional benefit to breast cancer patients. Citation Format: Claire Crafter, Jon Curwen, Oona Delpuech, Lenka Oplustilova, Stephen Green, Henry Brown, Cath Trigwell, Sabina Cosulich. Targeting interdependent signaling pathways to increase the durability and magnitude of response: promising combination therapy with dual mTORC1/2 inhibitors and CDK4/6 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4719. doi:10.1158/1538-7445.AM2015-4719

Abstract DDT01-03: Discovery and pre-clinical pharmacology of AZD9496: An oral, selective estrogen receptor down-regulator (SERD)

Abstract With over 70% of breast cancers expressing estrogen receptor alpha protein (ERα), treatment with either anti-hormonal therapies that directly block ERα function (e.g. tamoxifen) or therapies that block the production of estrogen itself (e.g. anastrozole) have proven to be effective treatments for the disease. Following the discovery of the ERα antagonist tamoxifen in the 1960s, identification of the selective estrogen receptor down-regulator (SERD) fulvestrant represented a further step forward in the treatment of advanced ER+ breast cancer, especially in the endocrine resistance setting where ERα appears to be activated by a ligand independent route through other growth factor signaling pathways. In addition, fulvestrant has also shown significant overall survival (OS) results in the FIRST trial comparing 500 mg fulvestrant with anastrozole in first line advanced ER+ve patients where the majority of patients had not received prior endocrine therapy. Given fulvestrant's low bioavailability following intramuscular injection and the levels of ERα protein in clinical samples after treatment, the question remains as to whether an agent that could achieve higher steady state levels of drug more rapidly and drive further decreases in ERα levels would give enhanced clinical benefit. We have identified a novel, potent, non-steroidal SERD that can be administered orally and could yield improved exposure and clinical benefit. This presentation will describe the discovery and pre-clinical pharmacology of AZD9496, a small molecule that can antagonise ERα and induce receptor degradation in breast cancer cell lines at picomolar concentrations. The good oral pharmacokinetic properties of the compound in pre-clinical species led to significant tumor growth inhibition in an endocrine sensitive MCF-7 xenograft model at a dose of 5 mg/kg and >90% reduction in ER-regulated, progesterone receptor (PR) levels. Tumor regressions were seen in a long term estrogen deprived (LTED) in vivo model, representing the aromatase resistant setting, and corresponded with significant reductions in ERα protein levels, >90% at 5 mg/kg dose. AZD9496 also showed antagonist and down-regulation activity against ERα mutant protein both in vitro and in vivo. These findings strongly supported selection of AZD9496 as a clinical candidate for the treatment of ER+ve breast cancer and the drug is now under evaluation in a Phase 1 clinical trial. Citation Format: Hazel Weir, Mandy Lawson, Rowena Callis, Michael Hulse, Michael Tonge, Gareth Davies, Graeme Walker, Rachel Rowlinson, Jon Curwen, Zena Wilson, Steve Powell, Robert Bradbury, Alfred Rabow, Craig Donald, David Buttar, Richard Norman, Camila de Almeida, Peter Ballard, Gordon Currie, David Andrews, Graham Richmond, Anne Marie Mazzola, Ermira Pazolli, Brendon Ladd, Celina D'Cruz, Chris De Savi. Discovery and pre-clinical pharmacology of AZD9496: An oral, selective estrogen receptor down-regulator (SERD). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2015-DDT01-03

Synergistic antitumor effect of adenovirus armed with Drosophila melanogaster deoxyribonucleoside kinase and nucleoside analogs for human breast carcinoma in vitro and in vivo.

BackgroundSuicide gene therapy in cancer can selectively kill tumors without damaging normal tissues. Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK), an original suicide kinase, makes use of the carcinomatous suicide gene therapy for broader substrate specificity and a higher catalytic rate.MethodsTo enhance the anti-tumor efficacy of Dm-dNK and maintain its substrate specificity and safety control in the meantime, the conditionally replicative gene–viral system, ZD55–dNK (which contains the selective replication adenovirus, ZD55, encoded with Dm-dNK), was investigated in pushing a deeper development of this strategy. Selective replication, cell killing efficacy, and cytotoxicity, in combination with chemotherapy, were applied to two breast cell lines (MDA231 and MCF7 cells), two normal cell lines (WI38 and MRC5 cells), and the MCF7 xenograft model in vivo.ResultsThe preclinical study showed that ZD55–dNK, combined with 2′,2′-difluorodeoxycytidine (DFDC), synergistically inhibited adenovirus replication in vitro but maintained specifically cancer cell killing efficacy. ZD55–dNK also greatly improved the antineoplastic effect in vitro and in breast cancer xenograft in vivo.ConclusionThe concomitant use of ZD55–dNK and DFDC is possibly a novel and promising approach to breast cancer treatment, and further investigation on the safe control of excessive virus replication and the efficacy of this approach in humans is warranted.

Abstract 3906: Combined inhibition of PI3K isoforms and mTOR kinase is critical for cancer stem cell inhibition by VS-5584

Abstract Cancer stem cells (CSCs) represent a subpopulation of cancer cells that have tumor-initiating capability, are particularly resistant to chemotherapy, and can mediate tumor recurrence both locally and at metastatic sites. As such, these cells represent a critical challenge for effective treatment of cancer. High-throughput screening for small molecules that preferentially target CSCs identified dual inhibitors of the PI3K/mTOR pathway, suggesting the importance of this signaling pathway for CSC biology. Here we demonstrate that ablation of mTOR or individual PI3K isoforms by isoform-specific siRNA is not sufficient to reduce the proportion of CSCs. In contrast, combined knock down of PI3K isoforms and mTOR effectively reduced the proportion of CSCs in tumor cell lines. VS-5584 is a potent and selective pan-PI3K/TORC1/TORC2 inhibitor with equipotency against all four human Class I PI3K isoforms and the mTOR kinase. We demonstrate that VS-5584 preferentially targets CSCs in multiple orthogonal assays both in vitro and in human tumor xenograft models. Cancer stem cells express high levels of aldehyde dehydrogenase, and an Aldefluor assay that measures activity of this enzyme was used to identify CSCs. VS-5584 decreased the percentage of Aldefluor+ cells across multiple breast cancer cell lines. We demonstrated that VS-5584 preferentially induced apoptosis in Aldefluor+ SUM159 cells relative to Aldefluor- cells as measured by Annexin V and Caspase 3/7 assays. In contrast, paclitaxel induced more apoptosis in Aldefluor- than Aldefluor+ cells and enriched the percentage of CSCs. Another characteristic of CSCs is their enhanced ability to efflux cytotoxic agents. This drug-resistant CSC population, called side population (SP), can be monitored by exclusion of Hoechst dye. VS-5584 effectively eliminated the SP CSCs across multiple cancer types, while cisplatin and etoposide increased this subpopulation. Furthermore, ex vivo treatment of primary breast and ovarian tumor specimens with VS-5584 decreased the proportion of CSCs as measured by the Aldefluor assay and cell surface markers. Significantly, VS-5584 also targets CSCs in vivo in MDA-MB-231 triple negative and MCF7 ER+ breast cancer xenograft models as evidenced by decreases in the percentage of Aldefluor+ cells, tumorsphere-forming efficiency, and tumor-initiating capability in an in vivo limiting dilution re-implantation assay. Consistent with the notion that combined inhibition of PI3K isoforms and mTOR is critical for exerting a strong anti-CSC effect, the mTORC1-selective inhibitor everolimus did not reduce CSCs in the MCF7 xenograft model. The potent anti-CSC activities in primary patient cancer tissue and in xenograft models provide strong rationale for the clinical development of VS-5584 in combination with agents targeting the bulk tumor to potentially achieve durable clinical responses for cancer patients. Citation Format: Vihren N. Kolev, Quentin G. Wright, David T. Weaver, Jennifer E. Ring, Christian M. Vidal, Mahesh V. Pavdal, Jonathan A. Pachter, Qunli Xu. Combined inhibition of PI3K isoforms and mTOR kinase is critical for cancer stem cell inhibition by VS-5584. [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 3906. doi:10.1158/1538-7445.AM2014-3906

Optimization of 4-(N-Cycloamino)phenylquinazolines as a Novel Class of Tubulin-Polymerization Inhibitors Targeting the Colchicine Site

The 6-methoxy-1,2,3,4-tetrahydroquinoline moiety in prior leads 2-chloro- and 2-methyl-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)quinazoline (1a and 1b) was modified to produce 4-(N-cycloamino)quinazolines (4a–c and 5a–m). The new compounds were evaluated in cytotoxicity and tubulin inhibition assays, resulting in the discovery of new tubulin-polymerization inhibitors. 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin- 2(1H)-one (5f), the most potent compound, exhibited high in vitro cytotoxic activity (GI50 1.9–3.2 nM), significant potency against tubulin assembly (IC50 0.77 μM), and substantial inhibition of colchicine binding (99% at 5 μM). In mechanism studies, 5f caused cell arrest in G2/M phase, disrupted microtubule formation, and competed mostly at the colchicine site on tubulin. Compound 5f and N-methylated analogue 5g were evaluated in nude mouse MCF7 xenograft models to validate their antitumor activity. Compound 5g displayed significant in vivo activity (tumor inhibitory rate 51%) at a dose of 4 mg/kg without obvious toxicity, whereas 5f unexpectedly resulted in toxicity and death at the same dose.

An EGFR/CD13 bispecific fusion protein and its enediyne-energized analog show potent antitumor activity

Targeting to two or more objectives simultaneously has been pursued as a strategy to potentially increase the efficiency and selectivity of targeted drugs to certain cancers. In this study, an epidermal growth factor receptor (EGFR)/CD13-targeting, bispecific fusion protein ER(Fv)-LDP-NGR consisting of an anti-EGFR single-chain variable fragment (scFv), an apoprotein (LDP) of lidamycin (LDM), and a tri-CNGRC (Cys-Asn-Gly-Arg-Cys) peptide against CD13 was constructed, and then an enediyne-energized analog ER(Fv)-LDP-NGR-AE was generated by integration with an enediyne chomophore (AE) derived from LDM. The apoprotein LDP was used as a 'scaffold' to connect the scFv fragment and the tri-CNGRC peptide and also served as a specific 'carrier' for the extremely potent cytotoxic enediyne chromophore of LDM. Compared with its monospecific counterparts, ER(Fv)-LDP and LDP-NGR, the bispecific fusion protein ER(Fv)-LDP-NGR showed higher affinity to EGFR/CD13-overexpressed tumor cells. Determined by the MTT assay, the bispecific, enediyne-energized ER(Fv)-LDP-NGR-AE showed highly potent cytotoxicity to EGFR/CD13-overexpressed MCF-7 cells, with an IC50 value of 3.4×10 mol/l, whereas for the EGFR-overexpressed A431 cells, the IC50 value was 2.2×10 mol/l. For MCF-7 cells, the bispecific ER(Fv)-LDP-NGR-AE was more potent in cytotoxicity than the corresponding monospecific energized fusion proteins. In athymic mice models, the bispecific fusion protein ER(Fv)-LDP-NGR presented stronger inhibitory activity than the monospecific ER(Fv)-LDP and LDP-NGR. For the enediyne-energized fusion proteins, ER(Fv)-LDP-NGR-AE significantly inhibited the growth of EGFR/CD13-overexpressed MCF-7 xenograft and EGFR-overexpressed A431 xenograft by 86.3 and 81.4%, respectively. In addition, the bispecific ER(Fv)-LDP-NGR-AE showed much higher efficacy than its monospecific analogs ER(Fv)-LDP-AE and LDP-NGR-AE in both MCF-7 and A431 xenograft models. The results show that EGFR/CD13 bitargeting effectively improved the antitumor efficacy. Both the bispecific fusion protein and its enediyne-energized analog are highly effective in athymic mice bearing xenografts, and the latter exerts more marked efficacy. Generation of a pair of bispecific antibody-based therapeutics and its corresponding antibody-drug conjugate simultaneously may be a feasible strategy for the development of new targeted drugs for cancer therapy.

Abstract P4-15-02: The PI3K inhibitor GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models

Abstract The phosphoinositide 3-kinases (PI3Ks) are lipid kinases that activate the PI3K signaling pathway and play an essential role in regulating breast tumor cell growth, migration, and survival. Activating and transforming mutations in the PIK3CA gene (PI3K alpha) are commonly found in HER2+ breast cancer and ER+ breast cancer. GDC-0032 is a selective, orally bioavailable inhibitor with a Ki of 0.29 nM for PI3K alpha with 30 fold less inhibition of PI3K beta. In addition, GDC-0032 has increased single agent activity against PI3K alpha mutant and HER2 amplified cancer models in vitro and in vivo. The aim of our study was to evaluate the efficacy of GDC-0032 in breast cancer models in combination with standard of care therapeutics including taxanes, endocrine therapies, and HER2 targeted therapies. We evaluated cell viability for a range of dose concentrations of GDC-0032, as single agent and in combinations, in eleven breast cancer cell lines that harbor mutations in PI3K alpha (K111N, E545K, H1047R) and/or over-expressed HER2. GDC-0032 was combined with taxanes (paclitaxel and docetaxel), endocrine therapies (fulvestrant and tamoxifen) or anti-HER2 agents (trastuzumab, pertuzumab or ado-trastuzumab-emtansine) and synergy quantified using the Chou and Talalay method of Combination Index (C.I.). The combination of GDC-0032 with taxanes, endocrine therapies or anti-HER2 therapies were synergistic in the breast cancer cell lines tested based on C.I. values of less than 1.0. The combination of GDC-0032 with docetaxel in vitro resulted in decreased viability as a result of increased cell death. The in vitro results translated in vivo as GDC-0032 enhanced the anti-tumor activity of docetaxel and paclitaxel in MCF7 (E545K) xenografts that resulted in increased tumor regressions when GDC-0032 was dosed daily or intermittently on the same schedule as the taxane. In addition, the combination of GDC-0032 and fulvestrant or tamoxifen resulted in greater efficacy in the MCF7 xenograft model when compared to either agent alone. Enhanced efficacy was also observed when GDC-0032 was combined with trastuzumab or ado-trastuzumab-emtansine in the HER2+ BT474M1 (K111N) xenograft model. Moreover, the triple combinations of GDC-0032/trastuzumab/pertuzumab or GDC-0032/trastuzumab/docetaxel resulted in durable tumor regressions that were sustained in the HER2+ KPL-4 (H1047R) and BT474M1 xenograft models, respectively. All drug combinations with GDC-0032 were well tolerated in vivo based on minimal changes in body weight. Collectively, our preclinical data demonstrate that GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models and provides a strong rationale for further evaluation in patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-02.

Enzymatic preparation of κ-carrageenan oligosaccharides and their anti-angiogenic activity

The mixture of κ-neocarrabiose-sulfate, κ-neocarrahexaose-sulfate and κ-neocarraoctaose-sulfate were prepared from κ-carrageenan with enzyme. The anti-tumor and anti-angiogenic activity of obtained κ-carrageenan oligosaccharides (KOS), were explored. The results showed that KOS could inhibit the proliferation, migration and tube formation of ECV304 cells, and could inhibit the growth of new vessels in CAM model. KOS displayed strong anti-tumor activity in both S180 and MCF-7 xenograft models. Only human CD105 was detected in MCF-7 xenograft tumor, moreover KOS could decrease the growing of new blood vessels derived from tumor cell. Real-time PCR results showed that KOS could suppress the mRNA expression of human VEGF, bFGF, bFGFR and CD105 in MCF-7 xenograft tumor. All these results indicated that KOS has anti-tumor and anti-angiogenic activity in vivo and in vitro. Especially K has the potency to inhibit the differentiation of tumor cell to blood vessel endothelial cell.

Oxyphenisatin acetate (NSC 59687) triggers a cell starvation response leading to autophagy, mitochondrial dysfunction, and autocrine TNFα-mediated apoptosis.

Oxyphenisatin (3,3-bis(4-hydroxyphenyl)-1H-indol-2-one) and several structurally related molecules have been shown to have in vitro and in vivo antiproliferative activity. This study aims to confirm and extend mechanistic studies by focusing on oxyphenisatin acetate (OXY, NSC 59687), the pro-drug of oxyphenisatin. Results confirm that OXY inhibits the growth of the breast cancer cell lines MCF7, T47D, HS578T, and MDA-MB-468. This effect is associated with selective inhibition of translation accompanied by rapid phosphorylation of the nutrient sensing eukaryotic translation initiation factor 2α (eIF2α) kinases, GCN2 and PERK. This effect was paralleled by activation of AMP-activated protein kinase (AMPK) combined with reduced phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6K and 4E-BP1. Microarray analysis highlighted activation of pathways involved in apoptosis induction, autophagy, RNA/protein metabolism, starvation responses, and solute transport. Pathway inhibitor combination studies suggested a role for AMPK/mTOR signaling, de novo transcription and translation, reactive oxygen species (ROS)/glutathione metabolism, calcium homeostasis and plasma membrane Na+/K+/Ca2+ transport in activity. Further examination confirmed that OXY treatment was associated with autophagy, mitochondrial dysfunction, and ROS generation. Additionally, treatment was associated with activation of both intrinsic and extrinsic apoptotic pathways. In the estrogen receptor (ER) positive MCF7 and T47D cells, OXY induced TNFα expression and TNFR1 degradation, indicating autocrine receptor-mediated apoptosis in these lines. Lastly, in an MCF7 xenograft model, OXY delivered intraperitoneally inhibited tumor growth, accompanied by phosphorylation of eIF2α and degradation of TNFR1. These data suggest that OXY induces a multifaceted cell starvation response, which ultimately induces programmed cell death.The mechanistic basis for oxyphenisatin acetate anti-cancer activity remains unresolved. This study demonstrates that exposure is associated with an acute nutrient deprivation response leading to translation inhibition, induction of autophagy, transient estrogen receptor (ER) stress and mitochondrial dysfunction. Ultimately these effects promote apoptosis induction, which in ER+ breast cancer cells is mediated by autocrine TNFα production. This is the first study implicating a nutrient deprivation response as central to the downstream effects of oxyphenisatin acetate.

Estrone-3-Sulphate, a Potential Novel Ligand for Targeting Breast Cancers

The current study investigates the potential of estrone-3-sulphate (E3S) as a ligand for targeting Organic Anion Transporting Polypeptides (OATP), a family of membrane associated uptake transporters, for detection and diagnosis of hormone dependent breast cancers. E3S, an OATP substrate, is a predominant source of tumour estradiol in post-menopausal patients. To assess the potential of E3S as a ligand, distribution of exogenous E3S was determined at the whole body, tumour and cellular levels in murine models of hormone-dependent (MCF-7) and independent (MDA-MB-231) breast cancers. The highest levels of tumour uptake were observed at 6 h post injection (p.i) with significant difference (p = 0.04) between the level in MCF-7 (13.9±3.1%ID/g) and MDA-MB-231 (10.4±1.1%ID/g) (%ID/g: percentage of the total injected dose per gram tissue). The highest tumour-to-blood ratios (MCF-7∶7.4±1.2; MDA-MB-231∶9.1±2.1) were observed at 48 p.i., and highest tumour-to-muscle ratios (MCF-7∶10.7±1.5; MDA-MB-231∶3.8±0.7) were observed at 6 h p.i. Analogous to total tumour uptake, ex vivo tumour cell uptake at 2 h p.i. was 6 fold higher in MCF-7 in comparison to MDA-MB-231 tumour cells. Blocking studies, conducted by pre-administration of 100-fold excess E3S, resulted in significantly lower (MCF-7: p = 0.01; MDA-MB-231: p = 0.02) tumour uptake in both xenograft models, suggesting the involvement of an active carrier-mediated process. The expression of OATP1A2 was detected in tumour sections from both xenografts, with significantly higher expression (p = 0.002) in the MCF-7 xenografts. Overall, the higher tumour uptake and tumour-to-muscle ratio, alongside the higher expression of OATP1A2, in the MCF-7 xenograft model suggests the potential of E3S to serve as a novel ligand for targeting hormone dependent breast cancers.

Abstract 3929: Estrone-3-sulphate, a potential novel ligand for targeting breast cancers.

Abstract This study investigates the potential of estrone-3-sulphate (E3S) as a ligand for targeting Organic Anion Transporting Polypeptides (OATP), a family of membrane-associated transport proteins, for detection of hormone dependent breast cancers. E3S, a circulating inactive plasma estrogen, is an OATP substrate and a predominant source of tumour estradiol in post-menopausal patients. It has been reported that some of the OATP isoforms (e.g. OATP1A2, OATP2B1) are over expressed (up to ten times) in breast cancer tissues as compared to surrounding normal tissues. This suggests that the two- three times higher E3S concentration reported in malignant tissues, as compared to the surrounding normal tissues, could be due to higher expression and/or increased functional activity of OATPs detected in breast tumour tissues. We recently demonstrated in vitro that expression of some of the OATPs (i.e. OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, OATP3A1 and OATP4A1) were either exclusive, similar or significantly higher (p<0.001) in breast cancer cells as compared to normalized breast epithelial cells (MCF10A). Thus to assess in vivo, the potential of E3S as a ligand for targeting OATPs in breast cancers, distribution of exogenous E3S (administered intravenously) was determined in murine models of hormone-dependent (MCF-7) and independent (MDA-MB-231) breast cancers. The highest levels of tumour uptake were observed at 6h post injection (p.i) with significant difference between the level in MCF-7 (13.9 ± 3.1 %ID/g) and MDA-MB-231 (10.4 ± 1.1 %ID/g). The highest tumour-to-blood ratios (MCF-7: 7.4±1.2; MDA-MB-231: 9.1±2.1) were observed at 48 p.i., and highest tumour-to-muscle ratios (MCF-7: 10.7±1.5; MDA-MB-231: 3.8±0.7) were observed at 6h p.i. Analogous to total tumour uptake, ex vivo tumour cell uptake at 2h p.i. was 6 fold higher in MCF-7 compared to MDA-MB-231 tumour cells. Blocking studies, conducted by pre-administration of 100-fold excess E3S, resulted in significantly lower tumour uptake in both xenograft models, suggesting the involvement of an active carrier mediated process. Immunohistochemical analysis detected OATP1A2 in tumour sections from both xenografts, with significantly higher expression in the MCF-7 xenografts. Overall, the higher tumour uptake and tumour-to-muscle ratio, alongside the higher expression of OATP1A2, in the MCF-7 xenograft model suggest the potential of E3S to serve as a novel ligand for targeting hormone dependent breast cancers. To the best of our knowledge, this is the first report documenting the in vivo distribution of exogenous E3S, in models of hormone dependent and independent breast cancers. Citation Format: Nilasha Banerjee, Humphrey Fonge, Andrew Mikhail, Raymond M. Reilly, Reina Bendayan, Christine Allen. Estrone-3-sulphate, a potential novel ligand for targeting breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2013-3929

Investigational drug MLN0128, a novel TORC1/2 inhibitor, demonstrates potent oral antitumor activity in human breast cancer xenograft models

Aberrant activation of the mammalian target of rapamycin (mTOR) signaling plays an important role in breast cancer progression and represents a potential therapeutic target for breast cancer. In this study, we report the impact of the investigational drug MLN0128, a potent and selective small molecule active-site TORC1/2 kinase inhibitor, on tumor growth and metastasis using human breast cancer xenograft models. We assessed in vitro antiproliferative activity of MLN0128 in a panel of breast cancer cell lines. We next evaluated the impact of MLN0128 on tumor growth, angiogenesis and metastasis using mammary fat pad xenograft models of a non-VEGF (ML20) and a VEGF-driven (MV165) MCF-7 sublines harboring PIK3CA mutations. MLN0128 potently inhibited cell proliferation in various breast cancer cell lines harboring PIK3CA (IC(50): 1.5-53 nM), PTEN (IC(50): 1-149 nM), KRAS, and/or BRAF mutations (IC(50): 13-162 nM), and in human endothelial cells (IC(50): 33-40 nM) in vitro. In vivo, MLN0128 decreased primary tumor growth significantly in both non-VEGF (ML20; p = 0.05) and VEGF-driven MCF-7 (MV165; p = 0.014) xenograft models. MLN0128 decreased the phosphorylation of Akt, S6, 4E-BP1, and NDRG1 in both models. In contrast, rapamycin increased Akt activity and failed to reduce the phosphorylation of 4E-BP1, PRAS40, and NDRG1. VEGF-induced lung metastasis in MV165 is inhibited by MLN0128 and rapamycin. In conclusion, MLN0128 inhibits TORC1/2-dependent signaling in preclinical models of breast cancer. MLN0128 appears to be superior in blocking mTORC1/2 signaling in contrast to rapamycin. Our findings support the clinical research of MLN0128 in patients with breast cancer and metastasis.

The Functional Interaction between Acyl-CoA Synthetase 4, 5-Lipooxygenase and Cyclooxygenase-2 Controls Tumor Growth: A Novel Therapeutic Target

The acyl-CoA synthetase 4 (ACSL4), which esterify mainly arachidonic acid (AA) into acyl-CoA, is increased in breast, colon and hepatocellular carcinoma. The transfection of MCF-7 cells with ACSL4 cDNA transforms the cells into a highly aggressive phenotype and controls both lipooxygenase-5 (LOX-5) and cyclooxygenase-2 (COX-2) metabolism of AA, suggesting a causal role of ACSL4 in tumorigenesis. We hypothesized that ACSL4, LOX-5 and COX-2 may constitute potential therapeutic targets for the control of tumor growth. Therefore, the aim of this study was to use a tetracycline Tet-Off system of MCF-7 xenograft model of breast cancer to confirm the effect of ACSL4 overexpression on tumor growth in vivo. We also aim to determine whether a combinatorial inhibition of the ACSL4-LOX-COX-2 pathway affects tumor growth in vivo using a xenograft model based on MDA-MB-231 cells, a highly aggressive breast cancer cell line naturally overexpressing ACSL4. The first novel finding is that stable transfection of MCF-7 cells with ACSL4 using the tetracycline Tet-Off system of MCF-7 cells resulted in development of growing tumors when injected into nude mice. Tumor xenograft development measured in animals that received doxycycline resulted in tumor growth inhibition. The tumors presented marked nuclear polymorphism, high mitotic index and low expression of estrogen and progesterone receptor. These results demonstrate the transformational capacity of ACSL4 overexpression. We examined the effect of a combination of inhibitors of ACSL4, LOX-5 and COX-2 on MDA-MB-231 tumor xenografts. This treatment markedly reduced tumor growth in doses of these inhibitors that were otherwise ineffective when used alone, indicating a synergistic effect of the compounds. Our results suggest that these enzymes interact functionally and form an integrated system that operates in a concerted manner to regulate tumor growth and consequently may be potential therapeutic targets for the control of proliferation as well as metastatic potential of cancer cells.

Hwanggeumchal sorghum induces cell cycle arrest, and suppresses tumor growth and metastasis through Jak2/STAT pathways in breast cancer xenografts.

BackgroundCancer is one of the highly virulent diseases known to humankind with a high mortality rate. Breast cancer is the most common cancer in women worldwide. Sorghum is a principal cereal food in many parts of the world, and is critical in folk medicine of Asia and Africa. In the present study, we analyzed the effects of HSE in metastatic breast cancer.Methodology/Principal FindingsPreliminary studies conducted on MDA-MB 231 and MCF-7 xenograft models showed tumor growth suppression by HSE. Western blotting studies conducted both in vivo and in vitro to check the effect of HSE in Jak/STAT pathways. Anti-metastatic effects of HSE were confirmed using both MDA-MB 231 and MCF-7 metastatic animal models. These studies showed that HSE can modulate Jak/STAT pathways, and it hindered the STAT5b/IGF-1R and STAT3/VEGF pathways not only by down-regulating the expression of these signal molecules and but also by preventing their phosphorylation. The expression of angiogenic factors like VEGF, VEGF-R2 and cell cycle regulators like cyclin D, cyclin E, and pRb were found down-regulated by HSE. In addition, it also targets Brk, p53, and HIF-1α for anti-cancer effects. HSE induced G1 phase arrest and migration inhibition in MDA-MB 231 cells. The metastasis of breast cancer to the lungs also found blocked by HSE in the metastatic animal model.Conclusions/SignificanceUsage of HS as a dietary supplement is an inexpensive natural cancer therapy, without any side effects. We strongly recommend the use of HS as an edible therapeutic agent as it possesses tumor suppression, migration inhibition, and anti-metastatic effects on breast cancer.

Abstract 3781: Pharmacokinetics of endoxifen and tamoxifen in female mice: Implications for comparative in vivo activity studies

Abstract Background: In tamoxifen (TAM) treated patients, both CYP2D6 metabolism and low steady state concentrations of endoxifen (END), the active metabolite of TAM, have been associated with a higher risk of recurrence (Madlensky L, Clin Pharmacol Ther. 2011; 89(5):718-25). END drug development is ongoing at Mayo and NCI, and our previous data demonstrated that 1) END had high oral bioavailability in mice and yielded plasma concentrations and total exposure 20-fold greater as compared to an equivalent oral dose of TAM (MM Ames et al, Cancer Res 2011;70(8 Suppl 1):Abstract nr 3603) and 2) END is superior to TAM in both HER2 (Reinicke AACR 2011) and aromatase expressing (Goetz, SABC 2011) MCF-7 xenograft models. While subcutaneous (SQ) TAM is used by most researchers to study and develop models of TAM resistance, little is known about 1) whether murine and human TAM pharmacokinetics are comparable and 2) whether the route of administration affects concentrations of the active metabolites. Therefore, we conducted murine pharmacokinetic studies evaluating both the oral and SQ administration of TAM and END. Results: Following administration of TAM, concentrations of the active metabolites (END and 4-hydroxy TAM [4HT]) were therapeutic (Cmax- END 35.9 nM, 4HT 62.8 nM) only after a 0.5 mg oral dose of TAM. In contrast, following all doses of SQ TAM, concentrations of the active metabolites (END and 4HT) were sub-therapeutic (Cmax- END 5.2 nM; 4HT 18.7 nM). In contrast to humans, the Cmax of N-desmethyl TAM, the most abundant human TAM metabolite, was < 16.5 nM in mice after oral and SQ TAM. SQ administration of END and TAM resulted in substantially greater bioavailability of the parent drug compared with oral dosing. Following administration of 0.25-5 mg oral END and 0.0625-0.625 mg subcutaneous END, the increase in AUC was greater than dose proportional and active concentrations (>80 nM) were detected in plasma at every dose and both dosing routes. Comparable END and TAM plasma concentrations were achieved with a 0.0625 mg SQ END and a 0.5 mg SQ TAM, respectively. Conclusions: Our data strongly suggest that SQ dosing of TAM results in non-therapeutic concentrations of the active hydroxylated metabolites, and may be a simple pharmacokinetic factor influencing response and resistance in a murine in vivo xenograft model. Given the nonlinear pharmacokinetics and differences in metabolism, the dose and route of END must be carefully selected for comparative studies with TAM. These data support the ongoing human studies designed to bypass the need for TAM metabolism by the direct administration of END. Supported by the Mayo Comprehensive Cancer Center Grant (CA15083) and the Mayo Clinic Breast Cancer SPORE (CA 116201; MMA, JMR, MPG). 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 3781. doi:1538-7445.AM2012-3781

Akt and p53 are potential mediators of reduced mammary tumor growth by Chloroquine and the mTOR inhibitor RAD001

PI3K/Akt/mTOR and p53 signaling pathways are frequently deregulated in tumors. The anticancer drug RAD001 (everolimus) is a known mTOR-inhibitor, but mTOR-inhibition leads to phosphorylation of Akt inducing resistance against RAD001 treatment. There is growing evidence that conflicting signals transduced by the oncogene Akt and the tumorsuppressor p53 are integrated via negative feedback between the two pathways. We previously showed that the anti-malarial Chloroquine, a 4-alkylamino substituted quinoline, is a p53 activator and reduced the incidence of breast tumors in animal models. Additionally, Chloroquine is an effective chemosensitizer when used in combination with PI3K/Akt inhibitors but the mechanism is unknown. Therefore, our aim was to test, if Chloroquine could inhibit tumor growth and prevent RAD001-induced Akt activation. Chloroquine and RAD001 caused G1 cell cycle arrest in luminal MCF7 but not in mesenchymal MDA-MB-231 breast cancer cells, they significantly reduced MCF7 cell proliferation on a collagen matrix and mammospheroid formation. In a murine MCF7 xenograft model, combined treatment of Chloroquine and RAD001 significantly reduced mammary tumor growth by 4.6-fold ( p = 0.0002) compared to controls. Chloroquine and RAD001 inhibited phosphorylation of mTOR and its downstream target, S6K1. Furthermore, Chloroquine was able to block the RAD001-induced phosphorylation of Akt serine 473. The Chloroquine effect of overcoming the RAD001-induced activation of the oncogene Akt, as well as the promising antitumor activity in our mammary tumor animal model present Chloroquine as an interesting combination partner for the mTOR-inhibitor RAD001.

Structure-based design of thienobenzoxepin inhibitors of PI3-kinase

Starting from thienobenzopyran HTS hit 1, co-crystallization, molecular modeling and metabolic analysis were used to design potent and metabolically stable inhibitors of PI3-kinase. Compound 15 demonstrated PI3K pathway suppression in a mouse MCF7 xenograft model.

Abstract B146: XL388: A novel, selective, orally bioavailable mTORC1 and mTORC2 inhibitor that demonstrates pharmacodynamic and antitumor activity in multiple human cancer xenograft models

Abstract Background: The mTOR kinase exists in at least two distinct kinase complexes, mTORC1 and mTORC2 which collectively regulate cell growth, proliferation, survival, autophagy, and angiogenesis. Derivatives of the macrolide antibiotic rapamycin have shown encouraging clinical activity as cancer therapeutics; however, these molecules only partially inhibit mTORC1 and mTORC2 activity. Here we provide the first pre-clinical description of XL388, a selective and orally bioavailable small-molecule ATP-competitive inhibitor of mTORC1 and mTORC2 that demonstrates pharmacodynamic and anti-tumor activity in multiple human cancer xenograft models. Methods: The in vitro inhibition of mTORC1 and mTORC2 by XL388 was assayed using purified, recombinant mTOR/G L/raptor kinase and mTORC2 immunoprecipitated from cells, respectively. mTORC1 and mTORC2 substrate phosphorylation and pharmacodynamic activity were measured by phosphospecific immunoblot of lysates prepared from XL388-treated cancer cells or from xenograft tumor lysates prepared from XL388-treated animals. Cancer cell viability was determined by measuring cellular ATP concentrations or the disruption of cell membrane integrity. Synergy of XL388 with chemotherapeutics was measured by the combination index (CI) method as established by Chou and Talalay (1984). XL388 in vivo efficacy and pharmacodynamic studies were performed in nude mice bearing human tumor xenografts and dosed orally with XL388. Results and Conclusions: XL388 inhibits mTORC1 and mTORC2 in vitro with IC50 values of 8 nM and 166 nM, respectively. In MCF-7 cells, XL388 blocks mTORC1 phosphorylation of p70S6K (T389) with an IC50 value of 94 nM and blocks mTORC2 phosphorylation of AKT (S473) with an IC50 value of 350 nM. In vitro, XL388 inhibits the viability of solid and hematopoietic tumor cell lines when assayed as a single agent. XL388 also synergizes with chemotherapeutics in cell-based assays to block cell viability. When dosed orally once daily in mice, XL388 shows robust anti-tumor activity in multiple xenograft models including > 100% tumor growth inhibition in the MCF-7 xenograft model. Taken together, these data indicate that XL388 may have broad utility in cancer therapy both as a single agent and in combination with chemotherapeutics. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B146.

Effect of erythropoietin on human tumor growth in xenograft models

Recombinant human erythropoietin (rhEPO) has been used in the EU and the United States for the treatment of anemia in cancer patients after myelosuppressive chemotherapy or radiotherapy. However, several conflicting results have been reported concerning the detrimental effect of rhEPO on survival benefit in cancer patients. In experimental studies, contradictory results were also reported in in vitro tumor cell proliferation studies and in vivo tumor growth studies using tumor cells expressing EPO-receptor (EPO-R). Therefore, we tried to clarify the effect of epoetin β, a product of rhEPO, on tumor growth in xenograft models using five EPO-R-positive human cancer cell lines, namely the MCF7 breast, 786-O renal, SCH gastric, A549 lung and SK-OV-3 ovarian cancer cell lines. Epoetin β was administered once a week for 3 weeks at doses of 1,000, 3,000 and 10,000 IU/kg in accordance with the clinical administration schedule and dosages. As a result, no enhancement of tumor growth from the administration of epoetin β was observed in any of the xenograft models throughout the experiment duration. The effect of epoetin β on the antitumor activity of bevacizumab, an anti-angiogenic agent, was additionally examined using A549 and MCF7 xenograft models, since rhEPO reportedly stimulates tumor neovascularization. Epoetin β showed no significant effect on the antitumor activity of bevacizumab in either xenograft model. These findings suggest that epoetin β is not involved in in vivo tumor growth promotion.