Mice Bearing Breast Cancer Xenografts Research Articles

Efficient Intravenous Tumor Targeting Using the αvβ6 Integrin-Selective Precision Virotherapy Ad5NULL-A20.

: BackgroundWe previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvβ6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvβ6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvβ6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvβ6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvβ6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvβ6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvβ6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes.

Echinacoside Suppresses the Progression of Breast Cancer by Downregulating the Expression of miR-4306 and miR-4508.

The main treatment of breast cancer includes surgical resection, radiotherapy, chemotherapy, endocrine therapy, and molecular targeted therapy, but the outcomes remain unsatisfactory. Previous studies demonstrated that echinacoside, microRNA (miRNA/miR)-4306 and miR-4508 were associated with lymph node metastasis, chemoresistance and self-renewal capability in breast cancer, but in-depth studies on the underlying mechanism of their anticancer effects have not been performed to date. In order to identify the role of miR-4306 and miR-4508, and the mechanism of the antitumor effect of echinacoside in breast cancer, the present study first examined the expression of miR-4306 and miR-4508 in breast cancer tissues to examine their possible role in the development of breast cancer, then evaluated the effect of echinacoside on the expression of miR-4306 and miR-4508 on the viability, apoptosis, cell cycle, migration, and invasion abilities of breast cancer cells to explore the anti-cancer effect of echinacoside and the involvement of miR-4306 and miR-4508. Finally, the breast cancer cells and mice bearing breast cancer xenografts were treated with echinacoside and inhibitors of miR-4508 or miR-4306 to confirm their role on the anticancer effect of echinacoside. The results showed that miR-4306 and miR-4508 were decreased in breast cancer tissues and cells. Echinacoside inhibited cell proliferation, invasion and migration, and promoted the apoptosis of breast cancer cells by downregulating the expression of miR-4306 and miR-4508. In conclusion, this is the first study to show the association between echinacoside and miRNAs in cancer. The present study elucidates an underlying molecular mechanism of the antitumor effect of echinacoside on breast cancer, and thus may contribute to preventive and therapeutic strategies for breast cancer.

Untargeted Metabolomics Reveals Molecular Effects of Ketogenic Diet on Healthy and Tumor Xenograft Mouse Models.

The application of ketogenic diet (KD) (high fat/low carbohydrate/adequate protein) as an auxiliary cancer therapy is a field of growing attention. KD provides sufficient energy supply for healthy cells, while possibly impairing energy production in highly glycolytic tumor cells. Moreover, KD regulates insulin and tumor related growth factors (like insulin growth factor-1, IGF-1). In order to provide molecular evidence for the proposed additional inhibition of tumor growth when combining chemotherapy with KD, we applied untargeted quantitative metabolome analysis on a spontaneous breast cancer xenograft mouse model, using MDA-MB-468 cells. Healthy mice and mice bearing breast cancer xenografts and receiving cyclophosphamide chemotherapy were compared after treatment with control diet and KD. Metabolomic profiling was performed on plasma samples, applying high-performance liquid chromatography coupled to tandem mass spectrometry. Statistical analysis revealed metabolic fingerprints comprising numerous significantly regulated features in the group of mice bearing breast cancer. This fingerprint disappeared after treatment with KD, resulting in recovery to the metabolic status observed in healthy mice receiving control diet. Moreover, amino acid metabolism as well as fatty acid transport were found to be affected by both the tumor and the applied KD. Our results provide clear evidence of a significant molecular effect of adjuvant KD in the context of tumor growth inhibition and suggest additional mechanisms of tumor suppression beyond the proposed constrain in energy supply of tumor cells.

Polymer-lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells.

PurposeBreast cancer stem cells (CSCs) are responsible for the initiation, recurrence, and metastasis of breast cancer. Sufficient evidence has established that breast cancer cells can spontaneously turn into breast CSCs. Thus, it is essential to simultaneously target breast CSCs and cancer cells to maximize the efficacy of breast cancer therapy. HER2 has been found to be overexpressed in both breast CSCs and cancer cells. We developed salinomycin-loaded polymer–lipid hybrid anti-HER2 nanoparticles (Sali-NP-HER2) to target both HER2-positive breast CSCs and cancer cells.MethodsThe antitumor activity of Sali-NP-HER2 constructed by conjugating anti-HER2 antibodies to polymer–lipid salinomycin nanoparticles was evaluated in vitro and in vivo.ResultsSali-NP-HER2 efficiently bound to HER2-positive breast CSCs and cancer cells, resulting in enhanced cytotoxic effects compared with non-targeted nanoparticles or salinomycin. In mice bearing breast cancer xenografts, administration of Sali-NP-HER2 exhibited superior efficacy in inhibiting tumor growth. Sali-NP-HER2 reduced the breast tumorsphere formation rate and the proportion of breast CSCs more effectively than non-targeted nanoparticles or salinomycin alone.ConclusionSali-NP-HER2 represents a promising approach in treating HER2-positive breast cancer by targeting both breast CSCs and cancer cells.

Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix.

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR.

The PI3K/mTOR dual inhibitor P7170 demonstrates potent activity against endocrine-sensitive and endocrine-resistant ER+ breast cancer.

Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway has been implicated in anti-estrogen resistance in breast cancer. We tested the therapeutic potential of the novel PI3K/mTOR dual inhibitor P7170 in a panel of anti-estrogen-sensitive and anti-estrogen-resistant models of ER+ breast cancer. Estrogen receptor-positive (ER+) breast cancer cells were treated ±P7170. Fresh cores from primary ER+/HER2- tumors from two patients were treated ±P7170 ex vivo. Mice bearing breast cancer xenografts were randomized to treatment with vehicle, fulvestrant, P7170, or combinations, and tumor volumes were measured. Tissues and cells were analyzed for markers of pathway activity, cell viability, and apoptosis. In cell lines, P7170 exhibited IC50 values in the range of 0.9-7 nM and induced apoptosis. P7170 potently inhibited mTOR activity (≤ 25 nM) and inhibited PI3K at higher concentrations (≥ 200 nM). P7170 completely inhibited MCF-7 tumor growth, significantly inhibited growth of fulvestrant-resistant T47D tumors, and suppressed tumor cell proliferation but did not induce apoptosis. While P7170 inhibits PI3K and mTOR in ER+/HER2- human breast cancer cells and tumors ex vivo, in vivo data indicate that the primary mechanism of P7170 anti-tumor action is inhibition of mTOR and cell proliferation. P7170 is a novel agent worthy of further investigation for the treatment of ER+ breast cancer.

Recombinant GnRH-p53 protein sensitizes breast cancer cells to 5-fluorouracil-induced apoptosis in vitro and in vivo

An ideal approach to treat cancers with dysfunctional p53 tumor suppressor gene is to reinstate p53 functionality by directly using p53 protein as a therapeutic agent. However, this has not been possible because the cells cannot readily internalize the protein. We constructed a fusion protein consisting of gonadotropin-releasing hormone (GnRH-p53) and p53 moieties. The recombinant protein was directly used to treat human breast cancer cells and athymic nude mice bearing breast cancer xenografts, with or without DNA synthesis-arresting agent 5-fluorouracil (5-FU). Treatments of cells from breast cancer cell-lines MDA-MB-231, T47D, or SKBR-3 with GnRH-p53 in combination with 5-FU significantly enhanced p53-activated apoptosis signals, including PUMA expression, BAX translocation to mitochondria, and activated caspase-3. Intratumoral injection of the GnRH-p53 protein inhibited MDA-MB-231 xenograft growth and induced p53-mediated apoptosis in the tumors. Systemic treatment of the tumor-bearing mice via tail vein injection of GnRH-p53 markedly augmented the anticancer efficacy of 5-FU. Substitution of GnRH-p53 with wild type p53 protein had no effect. Recombinant GnRH-p53 is able to function as a surrogate of p53 with regard to its apoptosis-inducing activity. Combination of GnRH-p53 with DNA-damaging drugs may be of important therapeutic value for cancer treatment.

Susceptibility of HER2+ breast cancer cells to poly (ADP-ribose) polymerase (PARP) inhibition independent of an inherent DNA repair defect.

621 Background: HER2 overexpression in breast cancer confers increased tumor aggressiveness. Targeted therapy against HER2 has improved outcomes, but resistance and disease progression ultimately occurs, thus necessitating novel therapeutic strategies. PARP inhibitors target homologous recombination (HR) deficient tumors, such as the BRCA-associated breast and ovarian cancers. In this study, we report unexpected susceptibility of HER2+ breast cancer cells to PARP inhibition alone independent of an inherent HR deficiency. Methods: Cell viability was measured using colony formation and ATPLite assays. Tumor growth delay was assessed in vivo in mice bearing breast cancer xenografts. Proteins were detected by immunoblotting. HR was assayed using radiation (IR)-induced Rad51 foci or a GFP-based HR assay. NFκB activity was measured using a NFκB-driven luciferase assay. Results: Surprisingly, PARP inhibition with ABT-888 alone reduced the colony forming ability and cell viability of the HER2+ breast cancer cell lines BT474, SKBR3, MDA-MB361, and HCC1954 (~70 – 99% reduction at 10μM). This susceptibility did not correlate with an inherent HR deficit. Interestingly, HER2 overexpression itself may be one mechanism of susceptibility to ABT-888 as evidenced by increased cellular cytotoxicity and in vivo tumor growth delay of MCF7 cells stably expressing HER2. Further dissection of the mechanism revealed that NFκB transcriptional activity was significantly inhibited by ABT-888 (>95%) which corresponded with reduced levels of phosphorylated p65 and total IKKα, and a concomitant increase in IkBα. Furthermore, overexpression of p65 abrogated cellular sensitivity to ABT-888. Conclusions: HER2+ breast cancer cells are highly susceptible to PARP inhibition despite being HR proficient. This may be, in part, due to inhibition of NFκB. Further investigation of whether the addition of PARP inhibition to HER2 targeted therapy will delay the onset of resistance to therapy is warranted to potentially improve outcomes in HER2+ breast cancer patients.

Induction of tumor cell apoptosis by a novel class of N-thiolated β-lactam antibiotics with structural modifications at N1 and C3 of the lactam ring

The investigation of novel anti-tumor agents that preferentially select for malignant cells with a tolerable toxicity level has been the focus of anti-cancer drug discovery. Our laboratories have previously reported that certain N-alkylthiolated beta-lactams had DNA-damaging and apoptosis-inducing activity in various tumor lines but not in nontransformed cells. In the current study, we further delineated the effects of substitutions at C3 or N1 of the lactam ring for cell death-inducing capability with close attention paid to a discernible structure-activity relationship (SAR). We found that two beta-lactam analogs (JG-5 and JG-19), both containing a branched-chain moiety at C3 of the lactam ring, exhibit potent apoptosis-inducing activity. Additionally, JG-5 exhibited superior in vitro biological activity over JG-19 owing to structural modifications made to substituents at the N1 and C3 positions of the lactam ring. Furthermore, the branched beta-lactams were able to inhibit growth of mice bearing breast cancer xenografts, associated with induction of DNA damage and apoptosis in tumor tissues. Our results strongly warrant further investigation into these novel beta-lactams as potential anti-cancer therapeutics.

Pharmacodynamic (PD) endpoints and cellular proliferation effects of NVP-BEZ235, a dual PI3K/mTOR inhibitor, in breast cancer cells and xenografts with wild-type (WT) and activating PI3K mutations

14508 Background: PI3K mutations are frequent in human cancer and play a role in the malignant phenotype and therapeutic resistance. NVP-BEZ235 is a small molecular mass compound in phase I clinical studies. NVP-BEZ235 reversibly blocks the ATP binding pocket on both the p110 catalytic subunit of PI3Ks and mTOR. Our aims were to study the activity and PD markers of NVP-BEZ235 in breast cancer cell lines with activating PI3K mutations. Methods: A panel of breast cancer cell lines with either WT, endogenous PI3K activating mutations or over- expressing oncogenic p110α mutants were treated with NVP-BEZ235. A xenograft breast cancer model was used to determine in vivo effects. Results: Target inhibition by NVP-BEZ235 was achieved as evidenced by a reduction in the level of the phosphorylated form of Akt, S6 and 4EBP-1, plus the nuclear re-localisation of FoxO3a. Cellular proliferation in all tested cancer cell lines was strongly inhibited in the nanomolar range as determined by the growth inhibitory 50 concentration values. NVP-BEZ235 induced G1 cell cycle arrest and apoptosis in the breast cancer cell lines tested as determined by fluorescence-activated cell sorting and the presence of cleaved PARP. There was a strong correlation between the concentrations that inhibited signalling and those that induced a growth inhibitory effect. Importantly, NVP-BEZ235 was significantly active in cell cultures harbouring oncogenic PI3K mutants and demonstrated activity in models of trastuzumab resistance. Statistically significant antitumor activity was observed upon oral administration of NVP-BEZ235 to mice bearing breast cancer xenografts at tolerated doses. PD endpoints (p-Akt, p-4EBP1 and p-S6) in tumour tissue were reduced after compound treatment. Conclusions: NVP- BEZ235 shows a rapid and profound inhibition of the PI3K pathway and results in tumor cell growth inhibition and apoptosis. Disease stasis and inhibition of PD biomarkers was observed in breast cancer xenograft models. The observed correlation between the in vivo antitumor activity and the selected PD endpoints suggests that these markers may be of assistance in the dose-finding studies in patients. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Pharma AG Novartis Pharma AG

Monocyte Chemotactic Protein-1 Secreted by Primary Breast Tumors Stimulates Migration of Mesenchymal Stem Cells

Major barriers to effective adenovirus-based gene therapy include induction of an immune response and tumor-specific targeting of vectors. The use of mesenchymal stem cells (MSC) as systemic delivery vehicles for therapeutic genes has been proposed as a result of their combined ability to home in on the tumor site and evade the host immune response. This study is aimed at investigating factors mediating homing of human MSCs to breast cancer primary cultures and cell lines in vitro and in vivo. Fluorescently labeled MSCs were given to mice bearing breast cancer xenografts, and tumor tissue was harvested to detect MSC engraftment. MSC migration in response to primary breast tumors in vitro was quantified, and chemokines secreted by tumor cells were identified. The role of monocyte chemotactic protein-1 (MCP-1) in cell migration was investigated using antibodies and standards of the chemokine. Serum MCP-1 was measured in 125 breast cancer patients and 86 healthy controls. Engrafted MSCs were detected in metastatic breast tumors in mice after systemic administration. There was a significant increase in MSC migration in response to primary breast tumor cells in vitro (6-fold to 11-fold increase). Tumor explants secreted a variety of chemokines including GROalpha, MCP-1, and stromal cell-derived factor-1alpha. An MCP-1 antibody caused a significant decrease (37-42%) in MSC migration to tumors. Serum MCP-1 levels were significantly higher in postmenopausal breast cancer patients than age-matched controls (P < 0.05). These results highlight a role for tumor-secreted MCP-1 in stimulating MSC migration and support the potential of these cells as tumor-targeted delivery vehicles for therapeutic agents.

Destruction of breast cancers and their metastases by lytic peptide conjugates in vitro and in vivo

In a series of in vivo and in vitro experiments, the concept has been established that breast cancer cells that express LH/CG or LHRH receptors can be targeted and destroyed by constructs consisting of a lytic peptide moiety and a 15-amino acid segment of the β-chain of CG or by an LHRH lytic peptide conjugate. Data obtained in vitro established the validity of this concept, showed the specificities of the Hecate-βCG, and Phor14 and Phor21-βCG conjugates in killing cells that express functional LH/CG receptors and proved that the LH/CG receptor capacity is directly related to the compound's specificity. In in vivo experiments, Hecate-βCG, Phor14-βCG, and Phor21-βCG(ala) each caused highly significant reductions of tumor volume and tumor burden in nude mice bearing breast cancer xenografts; Hecate and Phor21 alone or conjugated with non-specific peptides were not effective. Most importantly, the lytic peptide conjugates were all highly effective in targeting and destroying disseminated breast cancer metastases in lymph nodes, bones, lungs and other organs.

A Nonlinear Model Predictive Control Algorithm for Breast Cancer Treatment

A nonlinear model predictive control (NMPC) algorithm was developed to dose the chemotherapeutic tamoxifen to mice bearing breast cancer xenografts. A novel saturating rate cell-cycle model (SCM) was developed to capture unperturbed tumor growth dynamics, and a bilinear tumor kill term was included in the G-phase to account for the cycle-specific nature of tamoxifen and its active metabolite. Drug pharmacokinetics were modeled using a three-compartment linear model, which successfully approximated parent compound and metabolite (4-hydroxytamoxifen) plasma concentrations as a function of time. Using daily tumor measurements, the model predictive control algorithm successfully reduced tumor volume along a specified reference trajectory over a period of 4 months. A more clinicallyrelevant implementation using weekly or biweekly tumor measurements, and a prediction horizon seven days beyond the measurement interval, also led to reduced tumor volumes. In the mismatch case, a controller based on the simpler linear cell-cycle model (LCM) was unable to track desired reductions in tumor volume. Controllers based on a lumped-parameter saturating Gompertz model (GM), however, can yield similar performance to those using the more complex saturating rate cell-cycle model. This performance was dependent on the cellcycle phase of drug effect, with poorer results for M-phase targeted drugs. Overall, NMPC is a suitable algorithm for the class of chemotherapy problems with daily drug dosing, and the algorithm developed here may be adaptable to the clinical setting for the treatment of human breast cancer patients.

Comparison of an 18F Labeled Derivative of Vasoactive Intestinal Peptide and 2-Deoxy-2-[ 18F]Fluoro-D-Glucose in Nude Mice Bearing Breast Cancer Xenografts

Purpose: A 18fluorine-labeled derivative of vasoactive intestinal peptide [ 18F- Arg 15 ,Arg 21 VIP( 18F-dVIP)] was evaluated as a potential imaging agent for breast cancer by comparison with 2-deoxy-2-[ 18F]fluoro-D-glucose (FDG) using standard ex vivo determinations and small animal position emission tomography (PET) imaging. Procedures: Human breast carcinomas, T-47D and MDA-MB231, tumor-bearing nude mice were injected intravenously with 18F-dVIP or FDG for imaging and/or biodistribution ( ex vivo) determined by gamma counting. Results: FDG had two- to three-fold greater tumor accumulation and target-to-non target contrast relative to 18F-dVIP. VIP receptors were detected in both tumor types but in low concentrations (<15,000 receptors/cell) consistent with lower uptakes. FDG was cleared rapidly from non-target tissues while 18F-dVIP cleared into the kidneys. Conclusions: 18F-dVIP uptake in mice T-47D tumors and kidneys determined by imaging correlated with values determined by ex vivo counting suggesting that tumor and other tissue uptakes can be quantified by in vivo positron projection imaging. (Mol Imag Biol 2002;4:369–379)

(Arg 15, Arg 21) VIP: Evaluation of Biological Activity and Localization to Breast Cancer Tumors

Moody, T. W., J. Leyton, E. Unsworth, C. John, L. Lang and W. C. Eckelman. (Arg 15, Arg 21) VIP: Evaluation of biological activity and localization to breast cancer tumors. Peptides 19(3) 585–592, 1998.—VIP analogs, which contain a single lysine amino acid, were synthesized and evaluated using breast cancer cells. (Arg 15, Arg 20) VIP, (Arg 15, Arg 21) VIP, and (Arg 20, Arg 21) VIP inhibited 125I-VIP binding to T47D cells with high affinity (IC 50 values of 1.2, 1.0, and 0.8 nM, respectively). The VIP analogs elevated cAMP in T47D cells with ED 50 values ranging from 0.1–1 nM. Because (Arg 15, Arg 21) VIP was the most potent at elevating cAMP, it was characterized further. (Arg 15, Arg 21) VIP transiently increased c-fos gene expression in breast cancer cells. N-Succinimidyl-4- 18F (fluoromethly) benzoate was prepared in one chemical step from N-succinimidyl-4-(4-nitrobenzenesulfonyl)oxomethyl)benzoate by adding 18F in acetone at room temperature. This prosthetic group was then reacted with (Arg 15, Arg 21) VIP ((RR) VIP). ( 18F-RR) VIP bound with high affinity to T47D cells and was rapidly internalized. ( 18F-RR) VIP was injected intravenously into nude mice bearing breast cancer xenografts and after 4 h, the density of ( 18F-RR) VIP was elevated in the tumors relative to normal organs. These data suggest that VIP receptors may be used to localize breast cancer tumors.