Cell-based Xenograft Models Research Articles

G-protein-coupled estrogen receptor 1 promotes peritoneal metastasis of gastric cancer through nicotinamide adenine dinucleotide kinase 1-mediated redox modulation.

Adipose tissue is the second most important site of estrogen production, where androgens are converted into estrogen by aromatase. While gastric cancer patients often develop adipocyte-rich peritoneal metastasis, the underlying mechanism remains unclear. In this study, we identified the G-protein-coupled estrogen receptor (GPER1) as a promoter of gastric cancer peritoneal metastasis. Functional invitro studies revealed that β-Estradiol (E2) or the GPER1 agonist G1 inhibited anoikis in gastric cancer cells. Additionally, genetic overexpression or knockout of GPER1 significantly inhibited or enhanced gastric cancer cell anoikis invitro and peritoneal metastasis invivo, respectively. Mechanically, GPER1 knockout disrupted the NADPH pool and increased reactive oxygen species (ROS) generation. Conversely, overexpression of GPER1 had the opposite effects. GPER1 suppressed nicotinamide adenine dinucleotide kinase 1(NADK1) ubiquitination and promoted its phosphorylation, which were responsible for the elevated expression of NADK1 at protein levels and activity, respectively. Moreover, genetic inhibition of NADK1 disrupted NADPH and redox homeostasis, leading to high levels of ROS and significant anoikis, which inhibited lung and peritoneal metastasis in cell-based xenograft models. In summary, our study suggests that inhibiting GPER1-mediated NADK1 activity and its ubiquitination may be a promising therapeutic strategy for peritoneal metastasis of gastric cancer.

Investigation of the role and mechanism of ARHGAP5-mediated colorectal cancer metastasis.

Background: Metastatic colorectal cancer (CRC) is a lethal disease; however, the underlying molecular mechanisms remain unclear and require further study.Methods: RNA-Seq, PCR, Western blotting, immunohistochemistry, ChIP and RNAi assays were performed to investigate Rho GTPase-activating protein 5 (ARHGAP5, aslo known as p190RhoGAP-B, p190-B) expression and the clinical relevance, functional roles and regulatory mechanisms of this protein using human CRC cells and tissues. In vivo, two cell-based xenograft models were used to evaluate the roles of ARHGAP5 in CRC metastasis.Results: Here, we report that ARHGAP5 expression is significantly increased in metastatic CRC tissues and is inversely associated with patient overall survival. The suppression of ARHGAP5 reduces CRC cell metastasis in vitro and in cell-based xenograft models. Furthermore, we show that ARHGAP5 promotes CRC cell epithelial-mesenchymal transition by negatively regulating RhoA activity. Mechanistically, cAMP response element-binding protein (CREB1) transcriptionally upregulates ARHGAP5 expression, and decreased miR-137 further contributes to ARHGAP5 mRNA stability in CRC.Conclusions: Overall, our study highlights the crucial function of ARHGAP5 in CRC metastasis, thus suggesting novel prognostic biomarkers and hypothetical therapeutic targets.

Systematic chemical screening identifies disulfiram as a repurposed drug that enhances sensitivity to cisplatin in bladder cancer: a summary of preclinical studies

BackgroundSince the standard gemcitabine and cisplatin (GC) chemotherapy for advanced bladder cancer yields limited therapeutic effect due to chemoresistance, it is a clinical challenge to enhance sensitivity to GC.MethodsWe performed high-throughput screening by using a library of known chemicals and repositionable drugs. A total of 2098 compounds were administered alone or with GC to human bladder cancer cells, and chemicals that enhanced GC effects were screened.ResultsDisulfiram (DSF), an anti-alcoholism drug, was identified as a candidate showing synergistic effects with cisplatin but not with gemcitabine in multiple cell lines. Co-administration of DSF with GC affected cellular localisation of a cisplatin efflux transporter ATP7A, increased DNA–platinum adducts and promoted apoptosis. Micellar DSF nanoparticles (DSF-NP) that stabilised DSF in vivo, enhanced the inhibitory effect of cisplatin in patient-derived and cell-based xenograft models without severe adverse effects. A drug susceptibility evaluation system by using cancer tissue-originated spheroid culture showed promise in identifying cases who would benefit from DSF with cisplatin.ConclusionsThe present study highlighted the advantage of drug repurposing to enhance the efficacy of anticancer chemotherapy. Repurposing of DSF to a chemotherapy sensitiser may provide additional efficacy with less expense by using an available drug with a well-characterised safety profile.

In vivo Efficacy Studies in Cell Line and Patient-derived Xenograft Mouse Models.

In vivo xenograft models derived from human cancer cells have been a gold standard for evaluating the genetic drivers of cancer and are valuable preclinical models for evaluating the efficacy of cancer therapeutics. Recently, patient-derived tumorgrafts from multiple tumor types have been developed and shown to more accurately recapitulate the molecular and histological heterogeneity of cancer. Here we detail the procedures for developing patient-derived xenograft models from breast cancer tissue, cell-based xenograft models, serial tumor transplantation, tumor measurement, and drug treatment.

Targeting Acute Myeloid Leukemia with a New CXCR4 Antagonist IgG1 Antibody (PF-06747143)in NOD/SCID Mice

Chemoresistance represents a considerable barrier to improving outcomes for patients with acute myeloid leukemia (AML) and therapeutic approaches using multiple lines of therapy have been unsuccessful as cancer cells acquire resistance to the chemotherapeutic agents to which they are exposed. This vulnerable patient group needs individualizing therapy through careful selection of appropriate agents based on specific signaling pathways. The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow microenvironment, is highly expressed in 25-30% of patients with AML and its expression is correlated with poor prognosis and drug resistance. The purpose of this study was to investigate a new humanized monoclonal IgG1 antibody to CXCR4 (PF-06747143) and its effects as a monotherapy in AML primary patient samples and in chemotherapy resistant patient-derived xenotransplantation (PDX) models. This antibody was previously shown to be able to induce cell death through its effector function (CDC and ADCC) and to be efficacious in cell-based xenograft models of AML, NHL, CLL and MM.Here we have shown that PF-06747143 binds strongly and specifically to AML cell lines and to AML primary cells, by flow cytometry. Of 16 samples evaluated, 7 displayed low CXCR4 expression (CXCR4neg/low) whereas 9 displayed high expression (CXCR4high). A good correlation was observed between 12G5, a commercially available CXCR4 Ab, and PF-06747143 staining, indicating that PF-06747143 can be used to stratify AML patients. Chemotaxis in response to CXCL12 was significantly inhibited in all AML patient primary samples analyzed. Administration of PF-06747143 to mice engrafted with AML patient cells (PDX models) induced rapid malignant cell mobilization into the peripheral blood at 4 hrs after a single antibody dose, with mobilized cell levels going back down to baseline at 24 hrs post-dose. This is in line with the ability of the antibody to block malignant cell homing to the bone marrow, inducing cell mobilization, as well as induction of cell death through effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different models: 1) P15 model characterized by high CXCR4 expression, inducing aggressive disease, with rapid progression of leukemia and widespread dissemination and chemoresistance; 2) P17 model characterized by a low CXCR4 expression, a less aggressive disease and limited dissemination. Weekly administration of PF-06747143 to leukemic mice previously engrafted with P17 or P15 malignant cells induced a sharp reduction of leukemia cells in the bone marrow, spleen and blood leading to increased survival of leukemic mice in both models. Activity of the antibody as monotherapy was superior to daunorubicin in the P15 chemoresistant model. Secondary transplantation of bone marrow cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143 treatment, with slower tumor growth in mice transplanted with PF-06747143-treated cells. In summary, PF-06747143, a CXCR4 IgG1 antibody, is significantly efficacious as a monotherapy and superior to daunorubicin in AML chemoresistant PDX models. These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 537: Is interrupting IGF1R signaling enough to overcome resistance to trastuzumab in HER2+ breast cancer models?.

Abstract Background: Trastuzumab resistance hampers its well-known efficacy in controlling HER2+ breast cancer progression. IGF1R is associated with worse outcomes in the HER2+ subtype of breast cancer (Br Cancer Res Treat 2012 132:131). Indeed, there is in vitro evidence showing that increased signaling through IGF1R confers resistance to HER2-targeting agents. However, the involvement of IGF1R and its downstream signaling pathways in this mechanism have yet to be confirmed. Purpose: Since, IGF1R is involved in the progression of breast cancers and resistance to trastuzumab therapy, co-suppression of this pathway with IGF1R mAB plus trastuzumab (T) or T-DM1 may, therefore, be efficacious in T-resistant breast cancer models. Methods: Here, we investigated the preclinical efficacy of IGF1R mAB (R7072) alone or in combination with T or T-DM1 in BT474/HerR (T-resistant) and MCF7/HER2-18 (HER2 stably overexpressed) cell lines. The inhibition of IGF1R pathway effectors was evaluated by Western blotting. Tumor growth inhibition after treatment of R7072, T/T-DM1 or the combination was examined in cell-based xenograft models. Results: 1) Doses as low as 10 μg/ml, R7072 abrogated p- AKT (S473 and T308) within 1hr of treatment in both MC7/HER2-18 and MCF7 cells (parental), 2) R7072 transiently blocked phosphorylation of downstream effector of mTOR, P70S6K (in 1hr treatment) in MCF7/HER2-18 cells but completely blocked of phosphorylation of P70S6K in MCF7 cells in both time points, 3) IGF1-induced AKT activation was inhibited in both HER2 overexpressed MCF7 and parental cells, however, both T and T-DM1 failed to block AKT activation following IGF1 stimulation in both cell lines, 4) in both cell lines, treatment of R7072 resulted in upregulation of p-ERK at higher time points, 5) R7072 was unable to block PI3K and its downstream effectors in HER2+/T-resistant cells, 6) in vivo xenograft data show that the combination of R7072 and T/T-DM1 has strongly enhanced antitumor effects in both MCF7/HER2-18 and BT474/HerR cells, but as a single agent, R7072 failed to block tumor growth in HER2+/T-resistant cells, and 5) similarly this combination significantly blocked the expression Ki67 and CD31 in tumor tissues. Conclusion: Our data suggest that the combination of R7072 and anti-HER2 antibody (T or T-DM1) is highly effective in the HER2+/T-resistant model and that similar level of inhibition cannot be achieved by either monotherapy. Citation Format: Yuliang Sun, Nandini Dey, Max Hasmann, Pradip De, Brian Leyland-Jones. Is interrupting IGF1R signaling enough to overcome resistance to trastuzumab in HER2+ breast cancer models?. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 537. doi:10.1158/1538-7445.AM2013-537 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Dual mTORC1/2 and HER2 Blockade Results in Antitumor Activity in Preclinical Models of Breast Cancer Resistant to Anti-HER2 Therapy

The PI3K/Akt/mTOR pathway is an attractive target in HER2-positive breast cancer that is refractory to anti-HER2 therapy. The hypothesis is that the suppression of this pathway results in sensitization to anti-HER2 agents. However, this combinatorial strategy has not been comprehensively tested in models of trastuzumab and lapatinib resistance. We analyzed in vitro cell viability and induction of apoptosis in five different cell lines resistant to trastuzumab and lapatinib. Inhibition of HER2/HER3 phosphorylation, PI3K/Akt/mTOR, and extracellular signal-regulated kinase (ERK) signaling pathways was evaluated by Western blotting. Tumor growth inhibition after treatment with lapatinib, INK-128, or the combination of both agents was evaluated in three different animal models: two cell-based xenograft models refractory to both trastuzumab and lapatinib and a xenograft derived from a patient who relapsed on trastuzumab-based therapy. The addition of lapatinib to INK-128 prevented both HER2 and HER3 phosphorylation induced by INK-128, resulting in inhibition of both PI3K/Akt/mTOR and ERK pathways. This dual blockade produced synergistic induction of cell death in five different HER2-positive cell lines resistant to trastuzumab and lapatinib. In vivo, both cell line-based and patient-derived xenografts showed exquisite sensitivity to the antitumor activity of the combination of lapatinib and INK-128, which resulted in durable tumor shrinkage and exhibited no signs of toxicity in these models. The simultaneous blockade of both PI3K/Akt/mTOR and ERK pathways obtained by combining lapatinib with INK-128 acts synergistically in inducing cell death and tumor regression in breast cancer models refractory to anti-HER2 therapy.

Abstract 1811: Antitumor activity of lapatinib in combination with the dual mTORC1/2 inhibitor INK-128 in breast tumors resistant to anti-HER2 therapy

Abstract Purpose: The PI3K/Akt/mTOR pathway is an attractive target in HER2 positive breast cancer refractory to anti-HER2 therapy. The hypothesis is that suppression of this pathway results in sensitization to anti-HER2 agents. However, this combinatorial strategy has not been comprehensively tested in models of trastuzumab and lapatinib resistance. Experimental Design: We analyzed in vitro cell viability and induction of apoptosis in five different cell lines resistant to trastuzumab and lapatinib. Inhibition of HER2/HER3 phosphorylation, PI3K/Akt/mTOR and ERK signaling pathways was evaluated by western blot. Tumor growth inhibition following treatment with lapatinib, INK-128 or the combination of both agents was evaluated in three different animal models: two cell-based xenograft models refractory to both trastuzumab and lapatinib and a xenograft derived from a patient who relapsed to trastuzumab-based therapy. Results: The addition of lapatinib to INK-128 prevented both HER2 and HER3 phosphorylation induced by INK-128, resulting in inhibition of both PI3K/Akt/mTOR and ERK pathways. This dual blockade synergistically enhanced cell death in five different HER2 positive cell lines resistant to trastuzumab and lapatinib. In vivo, both cell line-based and patient-derived xenografts showed exquisite sensitivity to the antitumor activity of the combination of lapatinib and INK-128, causing durable tumor shrinkage in the absence of any sign of toxicity. Conclusions: The simultaneous blockade of PI3K/Akt/mTOR and ERK pathways achieved by combining lapatinib with INK-128 acts synergistically in inducing cell death and tumor regression in breast cancer refractory to anti-HER2 therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1811. doi:1538-7445.AM2012-1811