In Vivo Validation of PAPSS1 (3'-phosphoadenosine 5'-phosphosulfate synthase 1) as a Cisplatin-sensitizing Therapeutic Target.

Purpose: Our previous screening efforts found that inhibition of PAPSS1 increases the potency of DNA-damaging agents in non-small cell lung cancer (NSCLC) cell lines. Here, we explored the clinical relevance of PAPSS1 and further investigated it as a therapeutic target in preclinical model systems.Experimental Design: PAPSS1 expression and cisplatin IC50 values were assessed in 52 lung adenocarcinoma cell lines. Effects of PAPSS1 inhibition on A549 cisplatin sensitivity under hypoxic and starvation conditions, in 3D spheroids, as well as in zebrafish and mouse xenografts, were evaluated. Finally, the association between PAPSS1 expression levels and survival in patients treated with standard chemotherapy was assessed.Results: Our results show a positive correlation between low PAPSS1 expression and increased cisplatin sensitivity in lung adenocarcinoma. In vitro, the potentiation effect was greatest when A549 cells were serum-starved under hypoxic conditions. When treated with low-dose cisplatin, PAPSS1-deficient A549 spheroids showed a 58% reduction in size compared with control cells. In vivo, PAPSS1 suppression and low-dose cisplatin treatment inhibited proliferation of lung tumor cells in zebrafish xenografts and significantly delayed development of subcutaneous tumors in mice. Clinical data suggest that NSCLC and ovarian cancer patients with low PAPSS1 expression survive longer following platinum-based chemotherapy.Conclusions: These results suggest that PAPSS1 inhibition enhances cisplatin activity in multiple preclinical model systems and that low PAPSS1 expression may serve as a biomarker for platin sensitivity in cancer patients. Developing strategies to target PAPSS1 activity in conjunction with platinum-based chemotherapy may offer an approach to improving treatment outcomes. Clin Cancer Res; 23(21); 6555-66. ©2017 AACR.

3'-Phosphoadenosine 5'-phosphosulfate synthase 1 (PAPSS1) knockdown sensitizes non-small cell lung cancer cells to DNA damaging agents.

Standard treatment for advanced non-small cell lung cancer (NSCLC) with no known driver mutation is platinum-based chemotherapy, which has a response rate of only 30-33%. Through an siRNA screen, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate PAPS, was identified as a novel platinum-sensitizing target in NSCLC cells. PAPSS1 knockdown in combination with low-dose (IC10) cisplatin reduces clonogenicity of NSCLC cells by 98.7% (p < 0.001), increases DNA damage, and induces G1/S phase cell cycle arrest and apoptosis. PAPSS1 silencing also sensitized NSCLC cells to other DNA crosslinking agents, radiation, and topoisomerase I inhibitors, but not topoisomerase II inhibitors. Chemo-sensitization was not observed in normal epithelial cells. Knocking out the PAPSS1 homolog did not sensitize yeast to cisplatin, suggesting that sulfate bioavailability for amino acid synthesis is not the cause of sensitization to DNA damaging agents. Rather, sensitization may be due to sulfation reactions involved in blocking the action of DNA damaging agents, facilitating DNA repair, promoting cancer cell survival under therapeutic stress or reducing the bioavailability of DNA damaging agents. Our study demonstrates for the first time that PAPSS1 could be targeted to improve the activity of multiple anticancer agents used to treat NSCLC.

Induction of autophagy is an early response to gefitinib and a potential therapeutic target in breast cancer.

Gefitinib (Iressa(®), ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p<0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p<0.05) in delaying tumor growth than either monotherapy (p>0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers.

Combined RNAi-mediated suppression of Rictor and EGFR resulted in complete tumor regression in an orthotopic glioblastoma tumor model.

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.

Validating the use of a luciferase labeled breast cancer cell line, MDA435LCC6, as a means to monitor tumor progression and to assess the therapeutic activity of an established anticancer drug, docetaxel (Dt) alone or in combination with the ILK inhibitor, QLT0267.

A significant issue in drug efficacy studies is animal study design. Here we hypothesize that when evaluating new or existing therapeutics for the treatment of cancer, the location of disease burden will influence drug efficacy. To study this, Female NCr nude mice were inoculated with luciferase-positive human breast cancer cells (LCC6WT-luc) orthotopically (o.t.), intraperitoneally (i.p.) or intracardiacly (i.c.) to create localized, ascites or disseminated disease, respectively. Tumor development was monitored using bioluminescence imaging. Docetaxel (Dt) pharmacokinetics and distribution to sites of tumor growth were determined. Disease progression was followed in animals treated with Dt alone and in combination with QLT0267, an Integrin Linked Kinase inhibitor. Tumor related morbidity was most rapid when cells were inoculated i.c., where disease progression was observed in brain, ovaries, adrenal glands, and lungs. Dt pharmacokinetics were comparable regardless of the model used (mean plasma AUC0-24 hrs 482.6 ng/ml*hr), however, Dt levels were lowest in those tissues developing disease following i.c. cell injection. Treatment with low dose Dt (5 mg/kg) increased overall survival and reduced tumor cell growth in all three models but the activity was greatest in mice with orthotopic tumors. Higher doses of Dt (15 mg/kg) was able to prolong survival in animals bearing i.p. tumors but not i.c. tumors. Addition of QLT0267 provided no added benefit above Dt alone in the disseminated model. These studies highlight a need for more comprehensive in vivo efficacy studies designed to assess multiple disease models and multiple endpoints, focusing analysis of drug parameters on the most chemoresistant disease.

QLT0267, a small molecule inhibitor targeting integrin-linked kinase (ILK), and docetaxel can combine to produce synergistic interactions linked to enhanced cytotoxicity, reductions in P-AKT levels, altered F-actin architecture and improved treatment outcomes in an orthotopic breast cancer model.

INTRODUCTION: Substantial preclinical evidence has indicated that inhibition of integrin linked-kinase (ILK) correlates with cytotoxic/cytostatic cellular effects, delayed tumor growth in animal models of cancer, and inhibition of angiogenesis. Widely anticipated to represent a very promising therapeutic target in several cancer indications, it is increasingly evident that optimal therapeutic benefits obtained using ILK targeting strategies will only be achieved in combination settings. The purpose of this study was to investigate the therapeutic potential of the ILK small molecule inhibitor, QLT0267 (267), alone or in combination with chemotherapies commonly used to treat breast cancer patients. METHODS: A single end-point metabolic assay was used as an initial screen for 267 interactions with selected chemotherapeutic agents. These in vitro assays were completed with seven breast cancer cell lines including several which over-expressed human epidermal growth factor receptor 2 (Her2). One agent, docetaxel (Dt), consistently produced synergistic interactions when combined with 267. Dt/267 interactions were further characterized by measuring therapeutic endpoints linked to phosphorylated protein kinase B (P-AKT) suppression, inhibition of vascular endothelial growth factor (VEGF) secretion and changes in cytoarchitecture. In vivo efficacy studies were completed in mice bearing orthotopic xenografts where tumor growth was assessed by bioluminescence and calliper methods. RESULTS: The combination of 267 and Dt resulted in increased cytotoxic activity, as determined using an assay of metabolic activity. Combinations of cisplatin, doxorubicin, vinorelbine, paclitaxel, and trastuzumab produced antagonistic interactions. Further endpoint analysis in cell lines with low Her2 levels revealed that the 267/Dt combinations resulted in: a three-fold decrease in concentration (dose) of 267 required to achieve 50% inhibition of P-AKT; and a dramatic disruption of normal filamentous-actin cellular architecture. In contrast to Her2-positive cell lines, three-fold higher concentrations of 267 were required to achieve 50% inhibition of P-AKT when the drug was used in combination with Dt. In vivo studies focusing on low Her2-expressing breast cancer cells (LCC6) implanted orthotopically demonstrated that treatment with 267/Dt engendered improved therapeutic effects when compared with mice treated with either agent alone. CONCLUSIONS: The findings indicate that the 267/Dt drug combination confers increased (synergistic) therapeutic efficacy towards human breast cancer cells that express low levels of Her2.

Treatment with a cholesterol absorption inhibitor (FM-VP4) reduces body mass and adipose accumulation in developing and pre-obese mice.

OBJECTIVE: Disodium ascorbyl phytostanol phosphate (FM-VP4) is a cholesterol absorption inhibitor, a new class in cholesterol-lowering drug. Previous research on the lipid-lowering and anti-atherosclerotic effects of this drug has reported that administration of FM-VP4 results in a decrease in body mass. This study examined the FM-VP4 dose-dependent mass loss in mice and investigated some potential mechanisms by which decreased mass accumulation may have occurred. The effect of FM-VP4 administration on pre-obese mice was also tested. RESEARCH METHODS AND PROCEDURES: We conducted a dose-dependent study on mouse growth, food and water intake, organ mass, femur length, resting metabolic rate (RMR), maximal oxygen consumption under various conditions (VO(2swim) and VO(2heliox)), and fecal fat and plasma assessment for cholesterol and non-esterified fatty acids (NEFA) in mice fed a low fat (LF) or high fat (HF) diet, with or without FM-VP4. The ratio of lean to fat body mass of each animal was also assessed using magnetic resonance spectroscopy. To establish the effect of FM-VP4 on pre-existing obesity, mice were fed a high fat diet for 57 days, followed by administration of a diet containing 2% (w/w) FM-VP4 for 93 days. RESULTS: Animals exhibit a dose-dependent decline in body mass without a concomitant decrease in food intake, water intake, spleen, heart, or kidney mass, femur length or lean body mass. A dose-dependent trend toward a reduction in fat mass was observed in both high fat and low fat diet groups, becoming significant at a 1 and 2% FM-VP4 dosage (w/w). No FM-VP4 induced change in food or water intake, or resting metabolic rate was observed; however, an increase in VO(2swim) was observed in the 2% FM-VP4 group over HF control. These findings were also observed in the pre-obese group treated with 2% FM-VP4. DISCUSSION: We found a dose dependent reduction in mass accumulation in mice treated with FM-VP4. This loss of mass is not due to an increase in resting metabolic rate or decreased food or water intake. The only tissues exhibiting a decrease in mass with FM-VP4 treatment are liver and body fat. Fecal fat content increased significantly with FM-VP4 treatment in a dose-dependent manner, suggesting that the treatment reduces mass accumulation through decreased absorption or increased excretion of lipids.

Leishmania major abrogates gamma interferon-induced gene expression in human macrophages from a global perspective.

Infection with Leishmania major triggers several pathways in the host cell that are crucial to initial infection as well as those that are used by Leishmania to enhance its replication and virulence. To identify the molecular events of the host cell in response to Leishmania, the global gene expression of the human monocytic cell line THP-1 either infected with Leishmania major in the presence and absence of gamma interferon (IFN-gamma) or in the presence of IFN-gamma alone was analyzed using high-density human oligonucleotide microarrays, followed by statistical analysis. The persistence of the parasite despite an extensive response to IFN-gamma, added 24 h after infection with L. major, suggests that L. major can survive in an IFN-gamma-enriched environment in vitro. Results demonstrate that L. major counteracts the IFN-gamma response in macrophages on a large scale. Expression of genes involved in the innate immune response, cell adhesion, proteasomal degradation, Toll-like receptor expression, a variety of signaling molecules, and matrix metalloproteinases was significantly modulated.

Decreased levels of hypoxic cells in gefitinib treated ER+ HER-2 overexpressing MCF-7 breast cancer tumors are associated with hyperactivation of the mTOR pathway: therapeutic implications for combination therapy with rapamycin.

Developing novel synergistic and more effective combination treatments is necessary for better management of breast cancer in the clinic. It is established that HER-2 overexpressing breast cancers are sensitive to the HER-1 (epidermal growth factor receptor (EGFR)) inhibitor gefitinib, but that this targeted agent produces only moderate therapeutic effects in vivo. Here, we use a model of ER(+) HER-2 overexpressing MCF-7 breast cancer (MCF-7(HER-2)) to identify, as broadly as possible, the in vivo microenvironmental and molecular therapeutic responses to gefitinib to predict a therapeutically viable target for gefitinib-based combination treatment. Our data show a link between in vivo reductions in tumor hypoxia (3-fold decrease, P = 0.002) and elevated activity of the mTOR pathway (3.8-fold increase in phospho-p70-S6K protein, P = 0.006) in gefitinib treated MCF-7(HER-2) tumors. Despite decreased levels of phosphorylated EGFR, HER-2 and Erk1/2 (P = 0.081, 0.005 and 0.034, respectively) the expression of phospho-AKT was not reduced in MCF-7(HER-2) tumors after gefitinib treatment. Levels of ERalpha receptor were, however, 1.8-fold higher in gefitinib treated compared to control tumors (P = 0.008). Based on these results we predict that gefitinib activity against ER(+) HER-2 overexpressing EGFR co-expressing breast cancers should be enhanced if used with agents that target the mTOR pathway. In vitro studies using MCF-7(HER-2) and BT474 breast cancer cells exposed to gefitinib and rapamycin in combination show that this combination produced significantly greater growth inhibitory effects than either of the drugs alone. Chou and Talalay analysis of the data suggested that combination of gefitinib and rapamycin was synergistic (CI < 1) at a number of selected drug ratios and over a broad range of effective doses.

Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs.

Various methods have been explored to enhance antibody-based cancer therapy. The use of multivalent antibodies or fragments against tumor antigens has generated a great deal of interest, as various cellular signals, including induction of apoptosis, inhibition of cell growth/survival, or internalization of the surface molecules, can be triggered or enhanced on extensive cross-linking of the target/antibody complex by the multivalent form of the antibody. The goal of the studies reported here was to develop multivalent antibody constructs via grafting of antibody molecules onto liposome membranes to enhance antibody activity. Using trastuzumab and rituximab as examples, up to a 25-fold increase in the antibody potency in cell viability assay was observed when the antibodies were presented in the multivalent liposome formulation. Key cell survival signaling molecules, such as phosphorylated Akt and phosphorylated p65 nuclear factor-kappaB, were down-regulated on treatment with multivalent liposomal trastuzumab and liposomal rituximab, respectively. Potent in vivo antitumor activity was shown for liposomal trastuzumab. The data presented here showed the potential of liposome technology to enhance the therapeutic effect of antibodies via a mechanism that modulates cell survival through clustering of the target/antibody complex.

Development of a recombinant Leishmania major strain sensitive to ganciclovir and 5-fluorocytosine for use as a live vaccine challenge in clinical trials.

To provide a safer live challenge strain for use in clinical vaccine trials, a double drug sensitive strain of Leishmania major was derived using advances in gene targeting technology by stably introducing into the chromosome a modified HSV-1 thymidine kinase gene (tk), conferring increased sensitivity to ganciclovir (GCV), and a Saccharomyces cerevisiae cytosine deaminase gene (cd), conferring sensitivity to 5-fluorocytosine (5-FC). In vitro studies showed that the homozygous L. major (tk-cd+/+) promastigotes were killed by either drug alone, and together the drugs acted synergistically. In vivo infection studies showed that progressively growing lesions in BALB/c mice, caused by L. major (tk-cd+/+), were completely cured by 2 weeks of treatment with either drug alone or in combination. Treated animals showed no signs of reoccurrence of infection for at least 4 months when the experiments were terminated.

HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization.

The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6(HER-2)) into SCID-Rag2M mice. LCC6(HER-2) tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6(Vector)). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6(HER-2) versus LCC6(Vector) tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6(HER-2) tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content >/= 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6(HER-2) than in LCC6(Vector) tumors (16.41 +/- 8.1% and 5.96 +/- 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser(536)) nuclear factor-kappaB p65 were significantly elevated in LCC6(HER-2) tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1alpha protein levels was observed in LCC6(HER-2) compared with LCC6(Vector) tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6(HER-2) tumors, no significant differences were found (P > 0.05) between LCC6(HER-2) and LCC6(Vector) vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.

Treatment of HER-2/neu overexpressing breast cancer xenograft models with trastuzumab (Herceptin) and gefitinib (ZD1839): drug combination effects on tumor growth, HER-2/neu and epidermal growth factor receptor expression, and viable hypoxic cell fraction.

PURPOSE: The purpose of this research was to assess the effects of single agent and combination treatment with trastuzumab and gefitinib on tumor growth and tumor microenvironment in two HER-2/neu overexpressing breast xenograft models, MDA-MB-435/LCC6(HER-2) (LCC6(HER-2); estrogen receptor negative) and MCF-7(HER-2) (estrogen receptor positive). EXPERIMENTAL DESIGN: LCC6(HER-2) and MCF-7(HER-2) cells, both in tissue culture and xenografts grown in SCID-Rag 2M mice, were treated with trastuzumab and gefitinib, alone or in combination. The rate of tumor growth was determined. In addition, tumor HER-2/neu and epidermal growth factor receptor expression, cell viability, cell cycle distribution, and proportion of viable hypoxic cells were determined by flow cytometric analyses of single tumor cell suspensions. RESULTS: Both tumor models were very sensitive to trastuzumab and moderately sensitive to gefitinib in vivo. The combination resulted in therapeutic effects, as judged by inhibition of tumor growth, which was greater (albeit not statistically significant) than that observed with trastuzumab administered as a single agent. Trastuzumab was effective in down-regulating HER-2/neu, and gefitinib mediated a reduction in epidermal growth factor receptor expression on tumor cells. In LCC6(HER-2) tumors, trastuzumab significantly reduced tumor cell viability, which was not improved by the addition of gefitinib. Gefitinib dramatically reduced the proportion of viable hypoxic cells in LCC6(HER-2) and MCF-7(HER-2) tumors. This effect was abrogated by the addition of trastuzumab. CONCLUSIONS: Although in vivo efficacy studies in two HER-2/neu overexpressing breast xenograft models showed that the combination of trastuzumab and gefitinib was effective, analyses of various cellular parameters failed to reveal beneficial effects and argue that this drug combination may not be favorable.