Reduced Tumor Progression Research Articles

Abstract LB-080: The histone methyltransferase DOT1L promotes neuroblastoma by regulating gene transcription

Abstract Myc oncoproteins exert tumorigenic effects by regulating the expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation. DOT1L is the only known histone methyltransferase that catalyses H3K79 methylation. Here, we showed that N-Myc up-regulated DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. Knocking down DOT1L reduced mRNA and protein expression of the N-Myc target genes ODC1 and E2F2. DOT1L bound to the Myc Box II domain of N-Myc protein, and knocking down DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene-amplified neuroblastoma cells. In mice xenografted with neuroblastoma cells stably expressing doxycycline-inducible DOT1L small hair-pin RNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1 and E2F2 gene expression, and independently correlated with poor patient survival. Taken together, our data identify DOT1L as a novel co-factor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis, and DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Citation Format: Matthew Wong, Andrew Tee, Giorgio Milazzo, Jessica Bell, Stefan Hüttelmaier, Patsie Polly, Giovanni Perini, Christopher J. Scarlett, Tao Liu. The histone methyltransferase DOT1L promotes neuroblastoma by regulating gene transcription [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 LB-080. doi:10.1158/1538-7445.AM2017-LB-080

Abstract 1959: MTA1 promotes tumor progression and bone metastasis in prostate cancer via positive regulation of MUC1 and CTSB

Abstract About 80% of the time that prostate cancer cells metastasize, they spread to the bones. Metastasis involves activation of invasive programs through genetic and epigenetic alterations, including changes in expression of chromatin remodeler proteins. We have previously identified metastasis-associated protein 1 (MTA1), a chromatin remodeler, as a component of vicious cycle of bone metastasis and confirmed significantly higher expression of MTA1 in bone metastatic samples from patients. In an attempt to dissect molecular mechanisms of MTA1 action, we performed integrative analysis of our bone metastasis signature data along with MTA1 ChIP-Seq data, which revealed mucin 1 (MUC1) and cathepsin B (CTSB) as strong potential candidates responsible for MTA1-driven invasiveness.Here, we show that loss of function studies with MTA1 in PC3M cells exhibit reduced expression for MUC1 and CTSB. We also demonstrate that depletion of MTA1 in PC3M aggressive prostate cancer cellsdoes not affect cell proliferation but leads to decrease in colony forming ability, invasive and migratory property of these cells. When PC3M cells silenced for MTA1 and tagged with luciferase are used to generate subcutaneous or intracardiac xenografts, it leads to reduced tumor progression or decreased metastasis to bone, respectively as evident by bioluminescent measurements and histology of tumors. Ongoing experiments aim to validate the role of MUC1 and CTSB in MTA1-driven invasiveness and metastasis in prostate cancer. Citation Format: Avinash Kumar, Swati Dhar, Gisella Campanelli, Nasir A. Butt, Christian R. Gomez, Jason M. Schallheim, Anait S. Levenson. MTA1 promotes tumor progression and bone metastasis in prostate cancer via positive regulation of MUC1 and CTSB [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 1959. doi:10.1158/1538-7445.AM2017-1959

Abstract 2228: Altering rictor with diosmetin reduces tumor progression in orthotopic prostate cancer model

Abstract Rapamycin insensitive companion of mTOR (Rictor) is an essential subunit of mTOR complex 2, maintains the integrity of the complex and functions as full activator of Akt. Rictor has been implicated to be involved in growth and progression of malignancies. Reports suggested that overexpression of Rictor in prostate cancer tissues, which may have potential role in prostate cancer progression. We demonstrated silencing Rictor in PC-3 cells decreased p- Akt (Ser-473) expression; conversely p-Akt (Thr-308) and p-PKCα (Ser-657) expression was increasing. Additionally to observe the molecular mechanism of Rictor regulation, we took HAP1 cells (derived from male chronic myelogenous leukemia (CML) cell line KBM-7) were edited and silenced by CRISPR/Cas9 with 2 and 10 base pair Rictor in a coding exon 5. HAP1 haploid human cell line contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15; these cells are instrumental for genetic screening, as gene inactivation is greatly facilitated by the absence of a second gene copy. In these cells we observed p-Akt (Ser-473) expression was significantly less with more pronounced in 10 base pair than 2 base pair. Further to determine potential role of diosmetin (5, 7-Trihydroxy-4′-methoxyflavone; a natural flavonoid present in citrus plant, has anti-mutagenic properties) on Rictor silenced 2 base pair and 10 base pair HAP1 cells, we treated these cells in concentration response fashion, and observed decrease in p-Akt (Ser-473) expression than control cells. Next, we determine invivo potential of Rictor silencing, we used luciferase tag PC-3 cells silenced with Rictor gene orthotopically implanted in ventral prostate of mice. After 8 weeks of Rictor silenced cells implanted mice, we observed less luciferase activity in prostate and occasional metastasis than control luciferase PC-3 cells. Moreover, diosmetin fed mice in dose dependent fashion (20 and 50µg/animal/day) represented decrease in tumor luciferase activity with reduced tumor volume and no metastasis than control mice. We observed metastasis only in control and rapamycin (8 mg/kg body weight intraperitoneally every alternate day) treated mice in spine, lung and kidney. Taken together, our studies demonstrate that diosmetin fed animals resulted in growth inhibition and induction of apoptosis by altering Rictor signaling cascade. We are demonstrating that diosmetin modulates molecular targets viz., Rictor and Akt to alter cellular events and elicit anticancer effects in prostate cancer cells. Citation Format: Rebecca Pakradooni, Ahmad Khalifa, Riddhi Patel, Sanjeev Shukla. Altering rictor with diosmetin reduces tumor progression in orthotopic prostate cancer model [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 2228. doi:10.1158/1538-7445.AM2017-2228

AMPK β1 reduces tumor progression and improves survival in p53 null mice.

The AMP‐activated protein kinase (AMPK) is a heterotrimeric protein complex that is an important sensor of cellular energy status. Reduced expression of the AMPK β1 isoform has been linked to reduced survival in different cancers, but whether this accelerates tumor progression and the potential mechanism mediating these effects are not known. Furthermore, it is unknown whether AMPK β1 is implicated in tumorigenesis, and if so, what tissues may be most sensitive. In the current study, we find that in the absence of the tumor suppressor p53, germline genetic deletion of AMPK β1 accelerates the appearance of a T‐cell lymphoma that reduces lifespan compared to p53 deficiency alone. This increased tumorigenesis is linked to increases in interleukin‐1β (IL1β), reductions in acetyl‐CoA carboxylase (ACC) phosphorylation, and elevated lipogenesis. Collectively, these data indicate that reductions in the AMPK β1 subunit accelerate the development of T‐cell lymphoma, suggesting that therapies targeting this AMPK subunit or inhibiting lipogenesis may be effective for limiting the proliferation of p53‐mutant tumors.

Antitumoral effect of Ocoxin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is becoming one of the most prevalent types of cancer worldwide. The most efficient types of treatment at present include surgical resection and liver transplantation, but these treatments may only be used in a small percentage of patients. In order to identify novel therapeutic strategies for this disease, the present study explored the potential antitumoral effect of Ocoxin® oral solution (OOS) in HCC. OOS inhibited the proliferation of HCC cell lines in a time- and dose-dependent manner, being more efficient when used in combination with sorafenib, a standard of care treatment for patients diagnosed with advanced-stage disease. Mechanistic studies indicated that the effect of OOS was due to the induction of cell cycle arrest rather than the stimulation of apoptotic cell death. The cell cycle was slowed down in all phases in the HCC cell lines treated with OOS. Finally, when tested in animal models of HCC, OOS reduced tumor progression through the induction of necrosis in xenograft tumor models. Considering the poor prognosis and high resistance to antitumor treatments of HCC, the antiproliferative action of OOS, particularly in combination with sorafenib, provides the opportunity to investigate the effect of combined therapy in a clinical setting.

Tweety-Homolog 1 Drives Brain Colonization of Gliomas.

Early and progressive colonization of the healthy brain is one hallmark of diffuse gliomas, including glioblastomas. We recently discovered ultralong (>10 to hundreds of microns) membrane protrusions [tumor microtubes (TMs)] extended by glioma cells. TMs have been associated with the capacity of glioma cells to effectively invade the brain and proliferate. Moreover, TMs are also used by some tumor cells to interconnect to one large, resistant multicellular network. Here, we performed a correlative gene-expression microarray and in vivo imaging analysis, and identified novel molecular candidates for TM formation and function. Interestingly, these genes were previously linked to normal CNS development. One of the genes scoring highest in tests related to the outgrowth of TMs was tweety-homolog 1 (TTYH1), which was highly expressed in a fraction of TMs in mice and patients. Ttyh1 was confirmed to be a potent regulator of normal TM morphology and of TM-mediated tumor-cell invasion and proliferation. Glioma cells with one or two TMs were mainly responsible for effective brain colonization, and Ttyh1 downregulation particularly affected this cellular subtype, resulting in reduced tumor progression and prolonged survival of mice. The remaining Ttyh1-deficient tumor cells, however, had more interconnecting TMs, which were associated with increased radioresistance in those small tumors. These findings imply a cellular and molecular heterogeneity in gliomas regarding formation and function of distinct TM subtypes, with multiple parallels to neuronal development, and suggest that Ttyh1 might be a promising target to specifically reduce TM-associated brain colonization by glioma cells in patients.SIGNIFICANCE STATEMENT In this report, we identify tweety-homolog 1 (Ttyh1), a membrane protein linked to neuronal development, as a potent driver of tumor microtube (TM)-mediated brain colonization by glioma cells. Targeting of Ttyh1 effectively inhibited the formation of invasive TMs and glioma growth, but increased network formation by intercellular TMs, suggesting a functional and molecular heterogeneity of the recently discovered TMs with potential implications for future TM-targeting strategies.

Tris-base buffer: a promising new inhibitor for cancer progression and metastasis.

Neutralizing tumor external acidity with oral buffers has proven effective for the prevention and inhibition of metastasis in several cancer mouse models. Solid tumors are highly acidic as a result of high glycolysis combined with an inadequate blood supply. Our prior work has shown that sodium bicarbonate, imidazole, and free‐base (but not protonated) lysine are effective in reducing tumor progression and metastasis. However, a concern in translating these results to clinic has been the presence of counter ions and their potential undesirable side effects (e.g., hypernatremia). In this work, we investigate tris(hydroxymethyl)aminomethane, (THAM or Tris), a primary amine with no counter ion, for its effects on metastasis and progression in prostate and pancreatic cancer in vivo models using MRI and bioluminescence imaging. At an ad lib concentration of 200 mmol/L, Tris effectively inhibited metastasis in both models and furthermore led to a decrease in the expression of the major glucose transporter, GLUT‐1. Our results also showed that Tris–base buffer (pH 8.4) had no overt toxicity to C3H mice even at higher doses (400 mmol/L). In conclusion, we have developed a novel therapeutic approach to manipulate tumor extracellular pH (pHe) that could be readily adapted to a clinical trial.

Performance of Pyridylthiourea-Polyethylenimine Polyplex for siRNA-Mediated Liver Cancer Therapy in Cell Monolayer, Spheroid, and Tumor Xenograft Models.

Medical application of siRNAs relies on methods for delivering nucleic acids into the cytosol. Synthetic carriers, which assemble with nucleic acids into delivery systems, show promises for cancer therapy but efficiency remains to be improved. In here, the effectiveness of pyridylthiourea‐polyethylenimine (πPEI), a siRNA carrier that favors both polyplex disassembly and endosome rupture upon sensing the acidic endosomal environment, in 3 experimental models of hepatocellular cancer is tested. The πPEI‐assisted delivery of a siRNA targeting the polo‐like kinase 1 into Huh‐7 monolayer produces a 90% cell death via a demonstrated RNA interference mechanism. Incubation of polyplex with Huh‐7 spheroids leads to siRNA delivery into the superficial first cell layer and a 60% reduction in spheroid growth compared to untreated controls. Administration of polyplexes into mice bearing subcutaneous implanted Huh‐7Luc tumors results in a reduced tumor progression, similar to the one observed in the spheroid model. Altogether, these results support the in vivo use of synthetic and dedicated polymers for increasing siRNA‐mediated gene knockdown, and their clinical promise in cancer therapeutics.

Umbilical cord blood CD34+ progenitor-derived NK cells efficiently kill ovarian cancer spheroids and intraperitoneal tumors in NOD/SCID/IL2Rgnull mice

ABSTRACTAdoptive transfer of allogeneic natural killer (NK) cells is an attractive therapy approach against ovarian carcinoma. Here, we evaluated the potency of highly active NK cells derived from human CD34+ haematopoietic stem and progenitor cells (HSPC) to infiltrate and mediate killing of human ovarian cancer spheroids using an in vivo-like model system and mouse xenograft model. These CD56+Perforin+ HSPC-NK cells were generated under stroma-free conditions in the presence of StemRegenin-1, IL-15, and IL-12, and exerted efficient cytolytic activity and IFNγ production toward ovarian cancer monolayer cultures. Live-imaging confocal microscopy demonstrated that these HSPC-NK cells actively migrate, infiltrate, and mediate tumor cell killing in a three-dimensional multicellular ovarian cancer spheroid. Infiltration of up to 30% of total HSPC-NK cells within 8 h resulted in robust tumor spheroid destruction. Furthermore, intraperitoneal HSPC-NK cell infusions in NOD/SCID-IL2Rγnull (NSG) mice bearing ovarian carcinoma significantly reduced tumor progression. These findings demonstrate that highly functional HSPC-NK cells efficiently destruct ovarian carcinoma spheroids in vitro and kill intraperitoneal ovarian tumors in vivo, providing great promise for effective immunotherapy through intraperitoneal HSPC-NK cell adoptive transfer in ovarian carcinoma patients.

The Histone Methyltransferase DOT1L Promotes Neuroblastoma by Regulating Gene Transcription.

Myc oncoproteins exert tumorigenic effects by regulating expression of target oncogenes. Histone H3 lysine 79 (H3K79) methylation at Myc-responsive elements of target gene promoters is a strict prerequisite for Myc-induced transcriptional activation, and DOT1L is the only known histone methyltransferase that catalyzes H3K79 methylation. Here, we show that N-Myc upregulates DOT1L mRNA and protein expression by binding to the DOT1L gene promoter. shRNA-mediated depletion of DOT1L reduced mRNA and protein expression of N-Myc target genes ODC1 and E2F2 DOT1L bound to the Myc Box II domain of N-Myc protein, and knockdown of DOT1L reduced histone H3K79 methylation and N-Myc protein binding at the ODC1 and E2F2 gene promoters and reduced neuroblastoma cell proliferation. Treatment with the small-molecule DOT1L inhibitor SGC0946 reduced H3K79 methylation and proliferation of MYCN gene-amplified neuroblastoma cells. In mice xenografts of neuroblastoma cells stably expressing doxycycline-inducible DOT1L shRNA, ablating DOT1L expression with doxycycline significantly reduced ODC1 and E2F2 expression, reduced tumor progression, and improved overall survival. In addition, high levels of DOT1L gene expression in human neuroblastoma tissues correlated with high levels of MYCN, ODC1, and E2F2 gene expression and independently correlated with poor patient survival. Taken together, our results identify DOT1L as a novel cofactor in N-Myc-mediated transcriptional activation of target genes and neuroblastoma oncogenesis. Furthermore, they characterize DOT1L inhibitors as novel anticancer agents against MYCN-amplified neuroblastoma. Cancer Res; 77(9); 2522-33. ©2017 AACR.

Intratumoral delivery of bortezomib: impact on survival in an intracranial glioma tumor model.

OBJECTIVE Glioblastoma (GBM) is the most prevalent and the most aggressive of primary brain tumors. There is currently no effective treatment for this tumor. The proteasome inhibitor bortezomib is effective for a variety of tumors, but not for GBM. The authors' goal was to demonstrate that bortezomib can be effective in the orthotopic GBM murine model if the appropriate method of drug delivery is used. In this study the Alzet mini-osmotic pump was used to bring the drug directly to the tumor in the brain, circumventing the blood-brain barrier; thus making bortezomib an effective treatment for GBM. METHODS The 2 human glioma cell lines, U87 and U251, were labeled with luciferase and used in the subcutaneous and intracranial in vivo tumor models. Glioma cells were implanted subcutaneously into the right flank, or intracranially into the frontal cortex of athymic nude mice. Mice bearing intracranial glioma tumors were implanted with an Alzet mini-osmotic pump containing different doses of bortezomib. The Alzet pumps were introduced directly into the tumor bed in the brain. Survival was documented for mice with intracranial tumors. RESULTS Glioma cells were sensitive to bortezomib at nanomolar quantities in vitro. In the subcutaneous in vivo xenograft tumor model, bortezomib given intravenously was effective in reducing tumor progression. However, in the intracranial glioma model, bortezomib given systemically did not affect survival. By sharp contrast, animals treated with bortezomib intracranially at the tumor site exhibited significantly increased survival. CONCLUSIONS Bypassing the blood-brain barrier by using the osmotic pump resulted in an increase in the efficacy of bortezomib for the treatment of intracranial tumors. Thus, the intratumoral administration of bortezomib into the cranial cavity is an effective approach for glioma therapy.

Abstract A16: Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers

Abstract Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. We and others have shown that inhibition of the ATR checkpoint kinase is synthetically lethal with multiple distinct cancer-associated mutations, including p53 loss, oncogenic stress (HRAS-G12V, KRAS-G12D, NRAS-G12D, MYC and CCNE1), deficiency in homologous recombination (BRCA1/2, PALB2, ATM loss), alternative lengthening of telomeres (ALT), chromatin modification (SETD2 loss), and others. The specificity of ATR inhibitors is vital to their successful clinical application, since off-targeting increases toxicity to normal cells and limits value in personalized treatments. We report the generation of a novel class of highly potent and specific ATR inhibitors (ATRN series) that exhibit low nanomolar activity in cultured cells (IC50 = 2-8 nM) and do not detectably inhibit ATM, DNA-PK or mTOR (IC50 > 10 µM). In side-by-side comparisons, both the potency and selectivity of the ATRN series is superior to previously reported ATR inhibitors (VE821, VE822, AZD20, AZD6738, ETP-46464). In addition, the ATRN series has sufficient bioavailability and stability for in vivo application. Our lead compound (ATRN-119) slows progression of human BRCA2-deficient PDAC (CAPAN1) and RAS oncogene-driven p53-null colon tumors in mice with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived xenograft (PDX) ovarian tumors show a significant reduction in tumor progression after 5 weeks treatment of ATRN-119, and display no toxicity or significant weight loss. In addition, cancers that maintain or reacquire HR function, and thus are resistant to treatment with PARPi or cisplatin, remain responsive to ATRN series inhibitors. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian, and PARP resistant cancers, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies. In summary, the ATRN series is a highly selective and potent class of ATR inhibitors with therapeutic potential for treating a broad range of cancers. The potentially identified biomarkers, in particular HR-deficiency, will inform patient selection (a critical component for delivering medical benefit) for treatment with our agent and could reduce future clinical risk and side-effects. Thus targeting patients with HR-deficient cancers is a particularly promising strategy in treating a broad range of cancers. Citation Format: Laura R. Butler, Ryan L. Ragland, Hank J. Breslin, Erin George, Tina Gill, Matthew Scheiwer, Nicolas Gordon, Karen Knudsen, Fiona Simpkins, Oren Gilad and Eric J. Brown. Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A16.

Significance of lysil oxidase-like 2 (LOXL2) expression in hepatocellular carcinoma

Objective: In this study, we aimed to investigate the exact role of LOXL2 and the correlation between LOXL2 and epithelial-mesenchymal transition (EMT), and inhibition of LOXL2, using Beta-aminopropionitrile (BAPN), initiated reduced tumor progression in hepatocellular carcinoma (HCC). Methods: The expression levels of LOXL2 in HCC tissue and adjacent noncancerous tissue were evaluated by quantitative reverse transcription polymerase chain reaction and clionicopathological analysis. The effect of BAPN on cell proliferation, migration, and invasion was investigated in vitro. Additionally, LOXL2 expression in culture supernatant of HCC cell lines were measured. Results: Our study showed that LOXL2 expression was increased in HCC cell lines and tissue. There was a significant correlation between EMT status and LOXL2 levels (P = 0.004). BAPN reduced the migration and invasion of HCC cells. HCC patients with high expression of LOXL2 had relatively shorter disease-free survival (p = 0.009) and overall survival (p = 0.035). The expression levels of LOXL2 in culture supernatant were parallel to LOXL2 expression in HCC cell lines. Multivariate analysis demonstrated that portal vein invasion (p = 0.015), venous invasion (p = 0.026), serum AFP level (p = 0.019), and LOXL2 expression (p = 0.009), were independent prognostic factors. Conclusion: Our results suggest that increased LOXL2 expression may contribute to tumor progression and the clinical value of LOXL2 as a therapeutic target in HCC.

The angiotensin receptor blocker, Losartan, inhibits mammary tumor development and progression to invasive carcinoma

Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2−ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success.Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples.We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors.Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype.Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.

Abstract P6-12-17: H-ferritin allows nanometronomic treatment of breast cancer with doxorubicin preventing drug resistance and circumventing cardiotoxicity

Abstract Chemotherapeutic treatment of breast cancer is based on maximum tolerated dose (MTD) approach.1 However, advanced stage tumors are not effectively eradicated by MTD owing to suboptimal drug targeting, onset of therapeutic resistance and neoangiogenesis. In contrast, “metronomic” chemotherapy is based on frequent drug administrationsat lower doses, resulting in neovascularization inhibition and induction of tumor dormancy.1,2 However, several limiting factors remain for LDM in order to displace MTD treatments in clinical practice, including 1) low drug accumulation at tumor site,2 2) controversial effectiveness against chemoresistance in advanced metastatic cancers, and 3) acquired resistance after prolonged treatment. Recent advances in nanotechnology could offer groundbreaking solutions to improve the effectiveness of LDM chemotherapy, by taking advantage of the unique targeting efficiency of engineered nanocarriers.3 Here, we propose a new concept of “nanometronomic” chemotherapy, exploiting the H-ferritin (HFn)-mediated targeted delivery of doxorubicin (DOX)in an aggressive and metastatic breast cancer mouse model with DOX-inducible chemoresistance. HFn nanocages naturally target cancer cells4 owing to its affinity for transferrin receptor 1. HFn-DOX was recently demonstrated to overcome chemoresistance by actively promoting DOX nuclear translocation in vitro5,6 and was tested as a MTD treatment on a DOX-sensitive tumor model with encouraging results.7 We find that LDM administration of HFn-DOX strongly improves the antitumor potential of DOX chemotherapy arresting the tumor progression. Indeed, in vitro and in vivo results demonstrate that HFn nanocages mediate the nuclear delivery of DOX and increase DOX accumulation both in tumor tissue and in cancer cell nuclei, resulting in increased efficacy. Moreover, we find that HFn-DOX antitumor effect is attributable to multiple nanodrug actions beyond cell killing, including inhibition of tumor angiogenesis and avoidance of chemoresistance. Otherwise, although an even better reduction of tumor progression was achieved with liposomal DOX (pl-DOX) a five-fold increase in MDR-1-positive cells has been displayed, suggesting that liposomal DOX is not suitable in view of a protracted metronomic treatment, due to the onset of chemoresistance. Multiparametric assessment of heart tissues, including histology, ultrastructural analysis of tissue morphology, and measurement of markers of reactive oxygen species and hepatic/renal conditions, provided evidence that metronomic HFn-DOX allowed us to overcome cardiotoxicity contrary to what is observed with DOX and pl-DOX. Our results suggest that HFn-DOX has tremendous potential for the development of “nanometronomic” chemotherapy toward safe and tailored oncological treatments. 1. Kareva I, et al. Cancer Lett 2015; 358: 100. 2. Kerbel RS. Cancer Res Treat 2007; 39: 150. 3. Cruz-Munoz W, et al. Angiogenesis 2014; 7: 661. 4. Corsi F, Mazzucchelli S. Ther Deliv. 2016; 7: 149. 5. Bellini M, et al.. J Controlled Rel 2014;196: 184. 6. Zhang L, et al. Adv. Healthcare Mat. 2015; 4: 1305. 7. Liang M, et al. Proc Natl Acad Sci USA 2014; 111: 14900. Citation Format: Mazzucchelli S, Fiandra L, Bellini M, Truffi M, Rizzuto MA, Sorrentino L, Longhi E, Nebuloni M, Prosperi D, Corsi F. H-ferritin allows nanometronomic treatment of breast cancer with doxorubicin preventing drug resistance and circumventing cardiotoxicity [abstract]. In: Proceedings of the Thirty-Ninth Annual CTRC-AACR San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-17.

Abstract P3-05-07: Flutamide reduced tumor progression and altered steroid hormone secretion in human and canine inflammatory breast cancer cell lines

Abstract Background: Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate and accounts for 6% of diagnosed breast cancers. The role of androgens on breast cancer is on rise in research, trying to propose anti-androgen therapeutic strategies. The aim of this study was to determine the effects in vivo and in vitro of flutamide (anti-androgen drug) on cell proliferation, tumor progression and steroid production in two cancer IBC triple negative cell lines (SUM-149 and IPC-366, human and canine, respectively). Material and Methods: IPC-366 was cultured in Dulbecco's modified Eagle medium nutrient mixture F-12 Ham (DMEM/F12) and SUM149 was maintained in Ham's F-12 media. Flutamide concentrations added to the culture media were: 5 µm, 10 µm, and 15 µm for 72 hours. Additionally, IPC-366 and SUM149 xenotrasplanted mice were used for in vivo assays with the same flutamide concentrations administrated subcutaneously. Steroid hormones determination in culture media and tumor homogenates (pregnenolone (P5), progesterone (P4), dihydroepiandrostenedione (DHEA), androstenedione (A4), testosterone (T), dihydritestosterone (DHT), 17β-estradiol (E2) and estrone sulphate (SO4E1)) were assayed by EIA previously validated. Immunohistochemical (IHC) analysis of the steroidogenic enzymes CYP11A1, 3β-HSD, CYP19A1, 17β-HSD and 5α-reductase were assayed. Results: Percentage of cell proliferation showed a decrease in all treatments in IPC-366 and SUM149. In vivo tumor progression was reduced in around 65% in IPC-366 and SUM149 xenotrasplanted mice. Regarding hormonal secretion assayed in pellets and homogenates. in treated groups there was an increased in steroid secretion as showed the high levels found in P5, P4 and A4. T and DHT concentrations were higher in treated groups, in contrast to E2 levels that decreased. 17β-HSD and 5α-reductase by IHC showed a high expression in treated groups. Conclusion: IPC-366 and SUM149 treated with flutamide reduced the proliferation of neoplastic cells, reduced tumor progression in xenotrasplanted mice and altered steroid hormone secretion by increasing T production and decreasing in E2 levels. These results open a future approach for IBC and triple negative breast cancer. Citation Format: Caceres S, Pena L, Silvan G, Illera MJ, Monsalve B, Woodward WA, Reuben J, Illera JC. Flutamide reduced tumor progression and altered steroid hormone secretion in human and canine inflammatory breast cancer cell lines [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-07.

Liver Transplantation is a Preferable Alternative to Palliative Therapy for Selected Patients with Advanced Hepatocellular Carcinoma.

Patients with hepatocellular carcinoma (HCC) beyond the traditional criteria (advanced HCC) are typically offered palliation, which is associated with a 3-year survival rate lower than 30%. This study aimed to describe the outcomes for a subset of patients with advanced HCC who satisfied the Extended Toronto Criteria (ETC) and were listed for liver transplantation (LT). All patients listed in the Toronto liver transplantation program with HCC beyond both the Milan and University of California, San Francisco criteria were included in this study. Data were extracted from the prospectively collected electronic database. All radiologic images were reviewed by two independent radiologists. The primary end point was patient survival. Between January 1999 and August 2014, 96 patients with advanced HCC were listed for LT, and 62 (65%) of these patients received bridging therapy while on the waiting list. Bridging therapy led to a significant reduction in tumor progression (p=0.02) and tumor burden (p<0.001). The majority of those listed underwent LT (n=69, 72%). Both tumor progression on waiting list (hazard ratio [HR] 4.973; range1.599-15.464; p=0.006) and peak alpha-fetoprotein (AFP) at 400ng/ml or higher (HR, 4.604; range 1.660-12.768; p=0.003) were independently associated with waiting list dropout. Post-LT HCC recurrence occurredin 35% of the patients (n=24). Among those with HCC recurrence, survival was significantly better for those who received curative treatment (p=0.004). The overall actuarial survival rates from the listing were 76% at 1year, 56% at 3years, and 47% at 5years, and the corresponding ratesfrom LT were 93, 71, and 66%. Liver transplantation provides significantly better survival rates than palliation for patients with selected advanced HCC.

Novel Triazole-Piperazine Hybrid Molecules Induce Apoptosis via Activation of the Mitochondrial Pathway and Exhibit Antitumor Efficacy in Osteosarcoma Xenograft Nude Mice Model.

Mitochondria impart a crucial role in the regulation of programmed cell death and reactive oxygen species (ROS) generation, besides serving as a primary energy source. Mitochondria appeared as an important target for the therapy of cancer due to their significant contribution to cell survival and death. Here, we report the design and synthesis of a novel series of triazole-piperazine hybrids as potent anticancer agents. MCS-5 emerged as an excellent anticancer agent which showed better anticancer activity than the standard drug doxorubicin in in vitro and in vivo studies. MCS-5 displayed an IC50 value of 1.92 μM and induced apoptosis in Cal72 (human osteosarcoma cell line) cells by targeting the mitochondrial pathway. This compound arrested the G2/M phase of the cell cycle and induced ROS production and mitochondrial potential collapse in Cal72 cells. MCS-5 displayed excellent anticancer activity in the Cal72 xenograft nude mice model, where it significantly reduced tumor progression, leading to enhanced life span in treated animals compared to control and doxorubicin treated animals without exerting noticeable toxicity. In addition, a 2DG optical probe guided study clearly evoked that MCS-5 remarkably reduced tumor metastasis in the Cal72 xenograft nude mice model. These results indicate that MCS-5 appeared as a novel chemical entity which is endowed with excellent in vitro as well as in vivo anticancer activity and may contribute significantly to the management of cancer in the future.

Metformin enhancing the antitumor efficacy of carboplatin against Ehrlich solid carcinoma grown in diabetic mice: Effect on IGF-1 and tumoral expression of IGF-1 receptors

Diabetes has been listed as a risk factor for various types of cancer. Cancer cell development can be promoted by increased levels of IGF-1 and hyperinsulinemia that are associated with diabetes type II. Metformin is an anti-diabetic agent and its potential antitumor impact has become the objective of numerous studies. In this vein, we hypothesize that using metformin in diabetes type II mice may synergistic with carboplatin for reducing the risk of cancer. Therefore, the study aimed to evaluate the in vivo antitumor activity of metformin against solid EAC tumor growth in female diabetic mice and its potential pro-apoptotic and anti-proliferative effects with clarification of its inconclusive biological mechanisms. Mice were assigned into nine groups; normal control, diabetic control, diabetic plus EAC control, EAC control, and treated groups received carboplatin and/or metformin (100, 200mg/kg). Metformin administration especially with high dose potentiated the antitumor activity of carboplatin displayed by increased pro-apoptotic activators “caspase-3 and bax” and reduced anti-apoptotic protein bcl-2. This was confirmed by the histopathological scores. Moreover, the combination therapy was effective in attenuating the expression of the pro-angiogenic mediator “VEGF” and the microvessel density as revealed by the CD34. Additionally, this combination down-regulated the high levels of the mutagenic element “IGF-1” and its receptor expression, and attenuated the intensity of inflammatory mediators. In conclusion, it was found that metformin therapy could enhance apoptotic marker, and suppress the neovascularization and proliferation process. This clarified the ability of metformin to support carboplatin activity in reducing tumor progression in type II diabetes.

Oral administration of lyophilized Dunaliella salina, a carotenoid-rich marine alga, reduces tumor progression in mammary cancer induced rats.

Dunaliella salina is a photosynthetic cell factory used for the commercial production of food additives, cattle stock feed and cosmetics as well as active ingredients for pharmaceutical industries. The investigation of the in vivo antitumor activity of D. salina lyophilized powder (DSLP) against 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinogenesis in female Wistar rats indicated a dose-dependent effect of DSLP. We studied the effect of DSLP at two different dosages of 500 and 1000 mg per kg bw on DMBA induced mammary cancer in rats by measuring the status of antioxidant enzymes, phase I and phase II detoxification enzymes, lipid peroxidation, and glycoconjugated proteins and by investigating the expression pattern of cell proliferation (Ki-67), hormonal receptor (ER, PR and HER2) status by immunohistochemical analysis, and apoptotic (caspase-3 and -9) and pro-inflammatory (COX-2) markers by colorimetric analysis. After 16 weeks of the study, we observed 100% tumor formation (including high tumor incidence and tumor volume) and a significant increase in the level of hormonal receptors, cell proliferation, and pro-inflammatory and apoptosis markers in tumor-bearing animals compared to the control. The oral administration of DSLP (1000 mg per kg bw) to the DMBA treated animals showed up to 83.4% reduction of tumors and effectively restored the levels of biochemical markers in the mammary tissues in addition to the downregulation of the expression of molecular markers. In conclusion, DSLP was found to show a chemopreventive effect against breast cancer induced in rats through the suppression of cell proliferation and the induction of apoptosis.