Xenograft Tumor Tissues Research Articles

Fibroblast activation protein regulates tumor-associated fibroblasts and epithelial ovarian cancer cells

The fibroblast activation protein (FAP) is a cell surface serine protease which has emerged as a specific marker of tumor-associated fibroblasts (TAFs). FAP has been shown to have both invitro dipeptidyl peptidase and collagenase activity. However, the biological function of FAP in the tumor microenvironment is largely unknown. In this study, we first show that TAFs isolated from ovarian cancer samples have the characteristics of stem cells. To explore the functional role of FAP, the protein was silenced by siRNA lentiviral vector transfection. FAP silencing inhibited the growth of TAFs invitro, accompanied with cell cycle arrest at the G2 and Sphase in TAFs. FAP silencing also reduced the stem cell marker gene expression in TAFs. SKOV3 cells do not express FAP. Although FAP-silenced SKOV3 cells induced ovarian tumors, the rate of tumor growth was significantly decreased, as shown in the xenograft mouse model. TAF phenotypes in the xenograft tumor tissues were further assayed by immunohistochemistry. The expression of TAF markers, including fibroblast-specific protein, FAP, smooth muscle actin, desmin, vascular endothelial growth factor and fibroblast growth factor was decreased in the tumor stroma induced by FAP-silenced SKOV3 cells. In conclusion, FAP is an important regulator of the microenvironment in tumor formation and targeting FAP is a potential therapeutic strategy to combat ovarian cancer.

Differential response to EGFR- and VEGF-targeted therapies in patient-derived tumor tissue xenograft models of colon carcinoma and related metastases

Heterogeneity in primary tumors and related metastases may result in failure of antitumor therapies, particularly in targeted therapies for the treatment of cancer. In this study, patient-derived tumor tissue (PDTT) xenograft models of colon carcinoma with lymphatic and hepatic metastases were used to evaluate the response to EGFR- and VEGF-targeted therapies. Our results showed that primary colon carcinoma and its corresponding lymphatic and hepatic metastases have a different response rate to anti-EGFR (cetuximab) and anti-VEGF (bevacizumab) therapies. However, the underlying mechanism of these types of phenomenon is still unclear. To investigate whether such phenomena may result from the heterogeneity in primary colon carcinoma and related metastases, we compared the expression levels of cell signaling pathway proteins using immunohistochemical staining and western blotting, and the gene status of KRAS using pyrosequencing in the same primary colon carcinoma and its corresponding lymphatic and hepatic metastatic tissues which were used for establishing the PDTT xenograft models. Our results showed that the expression levels of EGFR, VEGF, Akt/pAkt, ERK/pERK, MAPK/pMAPK, and mTOR/pmTOR were different in primary colon carcinoma and matched lymphatic and hepatic metastases although the KRAS gene status in all cases was wild-type. Our results indicate that the heterogeneity in primary colon carcinoma and its corresponding lymphatic and hepatic metastases may result in differences in the response to dual-inhibition of EGFR and VEGF.

FKBP5 as a Selection Biomarker for Gemcitabine and Akt Inhibitors in Treatment of Pancreatic Cancer

We have recently shown that the immunophilin FKBP5 (also known as FKBP51) is a scaffolding protein that can enhance PHLPP-AKT interaction and facilitate PHLPP-mediated dephosphorylation of Akt Ser473, negatively regulating Akt activation in vitro. Therefore, FKBP5 might function as a tumor suppressor, and levels of FKBP5 would affect cell response to chemotherapy. In the current study, we have taken a step forward by using a pancreatic cancer xenograft mice model to show that down regulation of FKBP5 in shFKBP5 xenograft mice promotes tumor growth and resistance to gemcitabine, a phenomenon consistent with our previous findings in pancreatic cell lines. In addition, we also found that inhibitors targeting the Akt pathway, including PI3K inhibitor, Akt inhibitor and mTOR inhibitor had a different effect on sensitization to gemcitabine and other chemotherapeutic agents in cell lines, with a specific Akt inhibitor, triciribine, having the greatest sensitization effect. We then tested the hypothesis that addition of triciribine can sensitize gemcitabine treatment, especially in shFKBP5 pancreatic cancer xenograft mice. We found that combination treatment with gemcitabine and triciribine has a better effect on tumor inhibition than either drug alone (p<0.005) and that the inhibition effect is more significant in shFKBP5 xenograft mice than wt mice (p<0.05). These effects were correlated with level of Akt 473 phosphorylation as well as proliferation rate, as indicated by Ki67 staining in xenograft tumor tissues. These results provide evidence in support of future clinical trials designed to tailor therapy based on our observations.

Reversal effects of pantoprazole on multidrug resistance in human gastric adenocarcinoma cells by down-regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway in vitro and in vivo.

To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO(2) atmosphere at 37°C. Adriamycin (ADR)-resistant cells were cultured with addition of 0.8 µg/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK-8 Assay was performed to evaluate the cytotoxicity of anti-tumor drugs; BCECF-AM pH-sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H(+) -ATPases (V-ATPases), mTOR, HIF-1α, P-glycoprotein (P-gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 20 µg/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P < 0.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose-dependently (P < 0.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose-dependently. After 24-h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P < 0.05). The resistance index in PPZ pretreatment group was significantly lower than that in non-PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >10 µg/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P < 0.05). PPZ pretreatment also significantly inhibited protein expressions of V-ATPases, mTOR, HIF-1α, P-gp, and MRP1, and alter intracellular expressions in parent and ADR-resistant cells (P < 0.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti-tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti-tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway.

Abstract 958: PELP1 regulation of mTOR Axis: Role in breast cancer progression and resistance

Abstract Hormonal therapy resistance is a major clinical problem. Emerging evidence suggests that estrogen (ER) participates in extra-nuclear signaling events in the cytoplasm / membrane and such actions may play a role in therapy resistance. Recent studies suggested that mTOR pathway play a critical role in ER-mediated cell proliferation and growth factor signaling crosstalk. Proline, Glutamic-acid and Leucine-rich Protein 1 (PELP1) participates in ER extra-nuclear actions. PELP1 is a prognostic indicator of shorter breast cancer specific survival and PELP1 expression is predominantly in the cytoplasm in a subset of breast tumors. The objective of this application is to test whether cross talk occurs between mTOR-PELP1 signaling axis and whether mTOR targeting drugs can be used to target PELP1 oncogenic functions leading to therapy resistance. We have tested this hypothesis using ER-positive breast cancer cells that over express PELP1 (MCF7-PELP1, ZR75-PELP1) and models cells lacking PELP1 (MCF7-PELP1 shRNA, ZR-75-PELP1 shRNA). Vector transfected ZR75 and MCF7 cells were used as controls. Rapamycin and AZD8055 were used as pharmacological inhibitors to block mTOR pathway. Over expression of PELP1 enhanced the estrogen and Heregulin mediated cell proliferation in breast cancer cells. Rapamycin (10−7M) or AZD8055 (10−8M) treatment significantly reduced PELP1 driven growth in both MCF7 and ZR75 cells that are treated with estrogen. Similarly, Rapamycin and AZD8055 treatment of MCF7 and ZR75 cells significantly reduced PELP1 mediated increase in cell growth of Heregulin stimulated cells. Mechanistic studies using yeast two hybrid screen and reciprocal immunoprecipitations demonstrated that PELP1 directly interacts with mTOR and PP2A. PELP1cyto model cells that uniquely express PELP1 in the cytoplasm, exhibit excessive activation of mTOR signaling pathway upon estrogen and growth factor stimulation. Over expression of PELP1 in the breast cancer model cells increased phosphorylation of mTOR axis components (phos-mTOR, -S6K, -4EBP1,-AKT). Further, Knock down of PELP1 with siRNA significantly reduced the activation of mTOR signaling components upon estrogen and Heregulin stimulation. Immunohistochenistry studies using xenograft tumor tissues with PELP1 overexpression and PELP1cyto driven tumors with PELP1 deregulation showed correlation of PELP1 expression with the activation of mTOR signaling components and that PELP1cyto driven tumors have excessive activation of mTOR signaling components. Combination therapy of Tamoxifen and Rapamycin or AZD8055, sensitized PELP1 deregulated cells to hormonal therapy. Our results suggest that PELP1 driven oncogenic functions involve PELP1 modulation of mTOR signaling and blockade of mTOR signaling render PELP1 driven tumors highly sensitive to therapeutic inhibition. 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 958. doi:1538-7445.AM2012-958

Abstract 3724: Wogonin enhances tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo through H2O2-mediated downregulation of X-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis protein 1 and 2

Abstract Numerous malignant cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity, limiting value of TRAIL as an antitumor therapeutics. Combination of TRAIL with other agents is a promising strategy to overcome TRAIL resistance. Wogonin (5, 7-dihydroxy-8-methoxyflavone), a flavonoid originated from the root of the medicinal herb Scutellaria baicalensis Georgi, has been shown to enhance TRAIL-induced apoptosis in malignant cells in in vitro studies. However, whether wogonin enhances TRAIL's antitumor activity in vivo has never been studied. In this study, we first found that wogonin sensitized a variety of cancer cells including A549, HeLa and SKOV3 to TRAIL-induced apoptosis, as indicated by increased annexin V staining and enhanced activation of caspase-8 and -3 in TRAIL and wogonin cotreated cells, whereas having marginal effect on non-transformed bronchial epithelial cells and cervical epithelial cells. Then the effect of combination of TRAIL and wogonin was tested in a xenografted tumor model with A549 cells in nude mice. The results show that wogonin greatly enhanced TRAIL-induced suppression of tumor growth, which was accompanied with increased apoptosis in tumor tissues determined by a terminal deoxynucleotidyl-transferase -mediated dUTP nick-end labeling (TUNEL) assay. The protein levels of X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) were markedly reduced in both cultured cells and xenografted tumor tissues with co-treatment of wogonin and TRAIL. The down-regulation of these antiapoptotic proteins was likely mediated through proteasomal degradation and involved intracellular H2O2, as wogonin robustly induced H2O2 accumulation in A549 cells and the reactive oxygen species scavengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) and the proteasome inhibitor MG132 restored the expression of these antiapoptotic proteins and suppressed cytotoxicity in cells co-treated with wogonin and TRAIL. These results provide important in vivo evidence supporting the application of wogonin as TRAIL sensitizer for cancer 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 3724. doi:1538-7445.AM2012-3724

Abstract 5418: α-Mangostin, a dietary xanthone, inhibits ectopic xenograft growth of pancreatic cancer cells in an athymic nude mice

Abstract Pancreatic cancer (PC), the most aggressive malignant disease, ranks fourth most leading cause of cancer related death among men and woman in United States. Therefore, it is necessary to intensify our efforts for a better understanding of this disease and for the development of novel therapeutic strategies for its prevention and treatment. One approach to control this malignancy is to slow its progression through the use of natural agents present in the diet consumed by humans. α-Mangostin (MG), a xanthone constituent isolated from the pericarp of Mangosteen fruit (Garcinia mangostana), possesses various health promoting benefits including anti-oxidant, anti-inflammatory, anti-angiogenic and anti-cancer properties. We have previously shown that MG treatment induces apoptosis and inhibits growth of human PC cells via targeting NF-kB, Stat3 and Hedgehog signalings. Here, we determined the anti-tumor potential of MG against human PC in a xenograft mouse model. Highly aggressive human PC ASPC1 cells (2X106) were injected subcutaneously into the flanks of 14 athymic nude mice. Three days post-injection, mice were randomly divided into two groups. One group of mice was administered with MG (6 mg/kg body wt. i.p) in a 0.2 ml of PBS containing 25% polyethylene glycol (PEG) for 7 weeks. Vehicle control group mice were administered with 0.2 ml of PBS containing 25% PEG. Tumor volume of both group animals was measured and their body weight recorded weekly. All the mice of both groups were sacrificed when the tumor size of the control mice reached 1000 mm3. We observed that MG treatment inhibited the growth of ASPC1 cells in a xenograft mouse model as determined by significant (P&amp;lt;0.01) reduction in both the volume and weight of the tumors. Importantly, MG treatment inhibited angiogenesis as determined by the reduced number and size of blood capillaries at tumor sites. Vascular endothelial growth factor receptor (VEGF) is one of the components involved in tumor angiogenesis and metastasis. Immunohistochemistry (I.H.C) results demonstrated that MG treatment significantly reduced the expression of VEGF in xenograft tumors. Also, MG treatment inhibited markers of cell proliferation (ki67 and PCNA) in the xenograft tumor tissues as determined by I.H.C. No sign of toxicity was observed in any of the mice administered with MG as determined by histopathological examination of lungs, liver, kidneys and heart. One mouse from the control group showed metastatic foci of PC cells in the lungs but none of the MG treated animals showed any metastasis into any of the organs. These finding suggest that MG may reduce the metastatic burden of human PC. Taken together these results suggest that MG is a non-toxic dietary agent with anti-tumor potential against human PC. MG could be used as a novel dietary agent for the therapy of human PC. (Support: Department Pilot Project). 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 5418. doi:1538-7445.AM2012-5418

Abstract 4065: A simple biological imaging system for detecting adenovirus receptor expression in tumor cells using a telomerase-specific replication-competent adenovirus

Abstract Adenovirus serotype 5 (Ad5) is frequently used as an effective vector for induction of therapeutic transgenes in cancer gene therapy or of tumor cell lysis in oncolytic virotherapy. Ad5 can infect target cells through binding with the coxsackie and adenovirus receptor (CAR). Thus, the infectious ability of Ad5-based vectors depends on the CAR expression level in target cells. There are conventional methods to evaluate the CAR expression level in human target cells, including flow cytometry, western blotting and immunocytochemistry. We recently generated a green fluorescent protein (GFP)-expressing telomerase-specific replication-competent adenovirus OBP-401, which induces ectopic GFP expression in tumor cells, but not in normal cells. In this study, we evaluated whether induction of GFP expression by OBP-401 infection is associated with CAR expression in tumor cells. OBP-401-mediated GFP induction was further examined in xenograft tumor tissues that have different levels of CAR expression and in surrounding normal tissues. We used six human sarcoma cell lines that have different levels of CAR expression and CAR-positive normal human lung fibroblasts (NHLF). Flow cytometry analysis confirmed that cells of four of the sarcoma cell lines (OST, U2OS, NOS-10 and MNNG/HOS) as well as the NHLF cells showed detectable CAR expression, whereas cells of the NMFH-1 and OUMS-27 sarcoma cell lines had no detectable CAR expression. To determine suitable conditions for OBP-401 infection, all tumor and normal cells was infected with OBP-401 at multiplicity of infections of 1, 10 and 100 plaque forming units/cell. OBP-401 infection induced detectable GFP expression in CAR-expressing tumor cells, but not in CAR-negative tumor cells, nor in CAR-positive normal fibroblasts, 24 hours after infection. OBP-401-mediated GFP expression was significantly associated with CAR expression in tumor cells. OBP-401 infection detected tumor cells with low CAR expression more efficiently than conventional methods. We further examined the potential of the OBP-401-mediated method for the detection of CAR expression in tumor and normal tissues, we used this method to analyze CAR expression of human xenograft tumor tissues. OBP-401 infection induced GFP expression in OST tumor tissues, but not in OUMS-27 tumor tissues or in normal muscle tissue. OBP-401 also distinguished CAR-positive tumor tissues from CAR-negative tumor and normal tissues in biopsy samples. These results suggest that GFP-expressing telomerase-specific replication-competent adenovirus is a very potent diagnostic tool for assessment of functional CAR expression in tumor cells for Ad5-based antitumor 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 4065. doi:1538-7445.AM2012-4065

Abstract 1808: HM-032, a novel PI3K/mTOR dual inhibitor, is active on K-Ras/Raf mutation tumors through up-regulation of Bim

Abstract PIK3CA gene amplification and mutations have been identified in many types of tumors, suggesting the deregulation of PI3K/AKT/mTOR axis plays pivotal roles in tumorigenesis and development. K-Ras/Raf mutation is recognized as one of the key drug resistant factors for tumors against TKIs. HM-032 was reported here as a potent, selective and reversible ATP-competitive PI3K/mTOR dual inhibitor with special apoptotic activity in tumor cells harboring K-Ras mutations. HM-032 showed potent kinase inhibition with IC50 of 0.0008, 0.002, 0.001, 0.002 and 0.001 µM on PI3Kα, β, δ, ≤ and mTOR, respectively. In human prostate PC3 cells, it inhibited p-AKTS473, p-S6 and p-4EPB1 with IC50s of 0.002, 0.015 and 0.056 μM. Of the 89 tumor cell lines from lung, colon, breast, ovarian, and other tumor types, it was found that tumor cells with HER2 gene amplification, or PIK3CA mutation were relatively more sensitive to HM-032. Attractively, HM-032 showed apoptotic induction activity in the tumor cells harboring Ras/Raf mutations through inhibiting p-Erk and increasing gene expression of pro-apoptotic protein Bim. This result suggested that HM-032 might bring benefits to patients carrying K-Ras/Raf mutations in the clinic. Consistent with in vitro studies, HM-032 exhibited significant tumor growth inhibition in a dose dependent manner on multiple human xenograft models, including breast tumor MDA-MB-361(PIK3CAE545K, Her2 amplification), lung tumors H460 (PIK3CAE545K, K-RasQ61H) and A549 (LKB1 del, K-RasG12S), and glioblastoma U87MG (PTEN del) after 3 weeks oral dosing. Correspondingly, decrease of p-AKT and p-S6 was observed in the tumor xenograft tissues upon treatment with HM-032. In addition, HM-032 exhibited unique in vivo PK properties characterized by low clearance and long t1/2, which supported the intermittent dosing schedule in xenograft models. Based on the pre-clinical study results, HM-032 is anticipated to be a promising agent against solid tumors including carrying Ras/Raf mutations. 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 1808. doi:1538-7445.AM2012-1808

Abstract 2767: AUY922, a heat shock protein 90 inhibitor, plus 5-fluorouracil inhibits tumors synergistically in gastric cancer MKN45 xenograft via down regulation of multiple pivotal growth factor receptors and oncoproteins

Abstract Heat shock protein 90 (Hsp90) is the most abundant cellular chaperone and has nearly 100 pivotal client proteins, which are commonly dysregulated in cancers. The positive rate of Hsp90beta in gastric cancer (GC) tissue was approximately 30%, and was higher than non-cancerous gastric mucosa. Hsp90 inhibitors should have great potentials in the exploration for treatment strategy in gastric cancer (GC). NYP-AUY922-AG (AUY922), kindly provided by Novartis Pharma AG, is a novel resorcinylic isoxazole amide Hsp90 inhibitor. Using 72-hour MTT colorimetric assay, we have demonstrated that AUY922 has highly active growth inhibitory effects as a single agent of IC50 in the nanomolar ranges between 8.1 ± 0.2 and 14.7 ± 0.8 nM in the gastric cancer cell line panels (except c-met-overexpressing KATO-III cells with a IC50 of 158.1 ± 5.1 nM). In the gastric cancer MKN45 xenograft model, MKN45 cells (2 × 106) were injected subcutaneously in 100 αl of Matrigel/Hanks’ balanced salt solution to the flank of nude mice. AUY922 50 mg/kgw and 100 mg/kgw intraperitoneal weekly injection (i.p.) were used. Tumor inhibitory effects of AUY922 in the MKN45 human gastric cancer xenograft model were statistically evident in a dose-dependent and time-dependent manner. Several important growth factor receptors are overexpressed in GC, namely EGFR (epidermal growth factor receptor, HER1), HER-2/neu, and c-met (hepatocyte growth factor receptor, HGF receptor), etc. By immunohistochemical staining of xenograft tumor tissues using specific antibodies, AUY922 100 mg/kgw i.p. weekly significantly down regulates multiple growth factor receptors and oncoproteins, including c-met, HER-2/neu, IGF1-R, EGFR, Akt, and notably thymidylate synthase (TS), etc. While the effects of AUY922 50 mg/kgw are less statistically significant. Up to date, 5-fluorouracil (5-FU) or oral fluoropyrimidines are the current mainstay agents for first-line treatment in gastric cancer. By combining AUY922 100 mg/kgw i.p. and 5-fluorouracil (5-FU) 40 mg/kgw i.p. weekly (wk), tumor inhibitory effects of AUY922 and 5-FU were synergistically effective. Combining AUY922 50 mg/kgw or 100 mg/kgw i.p. weekly and other relevant chemotherapeutic agents has been also actively exploring. Our data indicate that AUY922 plus 5-fluorouracil (5-FU) inhibits tumors synergistically in gastric cancer MKN45 xenograft via down regulation of multiple pivotal growth factor receptors and oncoproteins. Since phase I study of AUY922 has been completed with favorable toxicity profiles, it is highly reasonable to combine AUY922 with 5-FU or other relevant chemotherapeutic agents in future development of clinical trials for gastric cancers. 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 2767. doi:1538-7445.AM2012-2767

Abstract 2519: U3-1565, a fully human anti-HB-EGF monoclonal antibody, inhibits oncogenic signaling and tumor cell growth in vitro and in vivo

Abstract Background: U3-1565 is a fully human monoclonal antibody directed against human heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family of ligands that bind to and activate the EGFR and HER4. HB-EGF-mediated activation of its target receptors results in oncogenic signaling. Methods: To determine inhibition of basal EGFR phosphorylation, human tumor cell lines were treated with U3-1565 or IgG2 and pEGFR levels were analyzed by ELISA. Inhibition of in vivo angiogenesis was analyzed by implanting HUVEC-based spheroids under the skin of SCID mice, which were treated with U3-1565, bevacizumab or PBS and analyzed for human neovasculature. To determine in vivo efficacy, SCID mice bearing EFO27-CL58 ovarian cancer xenografts were treated weekly with U3-1565, Cisplatin or with a combination of U3-1565 and Cisplatin. To perform xenograft analysis tumor-bearing mice were treated with U3-1565, cetuximab and erlotinib and primary xenograft tissue was collected, lyzed and pEGFR and pERK levels were analyzed by Western blotting. To determine expression of HB-EGF and pEGFR in human tumor tissue, Immunohistochemistry (IHC) stainings of human carcinoma samples were performed. Results: Inhibition of basal pEGFR levels by U3-1565 in comparison to control IgG2 treatment was demonstrated in various cancer cell lines. U3-1565 showed dose-dependent inhibition of HB-EGF-stimulated pHER4 and pERK activation in vitro (data not shown). Using a HUVEC-based spheroid in vivo system, inhibition of human endothelial cell sprouting by U3-1565 was shown. In a HB-EGF overexpressing ovarian cancer xenograft model U3-1565 demonstrated in vivo tumor cell growth inhibition as a single agent. Combination treatment of U3-1565 with Cisplatin resulted in tumor regression during the treatment phase and prevented re-growth of xenograft tumors after treatment stop. Analysis of EFO27-CL58 tumor xenograft tissue demonstrated strong reduction of pEGFR and partially reduced pERK levels after U3-1565 in vivo treatments. IHC staining of fresh human tumor samples revealed an overlapping expression pattern of HB-EGF and pEGFR. Conclusions: Our results demonstrate that U3-1565 inhibits basal activation of EGFR activation in vitro and reduces neo-vasculature formation (angiogenesis) in vivo. In a tumor xenograft model U3-1565 demonstrated tumor cell growth arrest as a single agent or in combination with Cisplatin or erlotinib. Analysis of xenograft material after in vivo exposure to U3-1565 strongly suggest that U3-1565's anti-tumor activity is based on inhibition of EGFR activation and prevention of oncogenic MAPK signaling. These data together with the evidence that human HB-EGF and activated EGFR are coexpressed in human tumor samples provide preclinical rational to develop U3-1565 as a clinical mAb candidate to treat cancer in human patients. 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 2519. doi:1538-7445.AM2012-2519

Establishment and characterization of a novel chordoma cell line: CH22

Chordoma is a rare primary malignant bone tumor and there exist only a few established human chordoma cell lines. The scarcity of robust chordoma cell lines has limited the ability to study this tumor. In this report, we describe the establishment of a novel chordoma cell line and characterize its in vitro and in vivo behaviors. The tumor tissue was isolated from a patient with recurrent chordoma of the sacrum. After 6 months in culture, the chordoma cell line, referred here as CH22, was established. Microscopic analysis of two-dimensional culture confirmed that the CH22 cells exhibited a typical vacuolated cytoplasm similar to the well-established chordoma cell line U-CH1. Electron microscopy showed cohesive cells with numerous surface filopodia, pockets of glycogen and aggregates of intermediate tonofilaments in cytoplasm. Three-dimensional culture revealed that the CH22 cells could grow and form clusters by day 8. The MTT assays demonstrated that, compared with sensitive osteosarcoma cell lines, CH22 cells were relatively resistant to conventional chemotherapeutic drugs. Western blotting and immunofluorescence analysis confirmed that the CH22 cells expressed brachyury, vimentin, and cytokeratin. Finally, histological analysis of CH22 xenograft tumor tissues demonstrated the appearance of physaliphorous cells and positive staining of brachyury, cytokeratin, and S100. By CT and MRI, imaging xenografts showed the typical appearances seen in human chordomas. These findings suggest that the established novel human chordoma cell line CH22 and its tumorigenecity in SCID nude mice may serve as an important model for studying chordoma cell biology and the development of new therapeutic modalities.

Antitumor effect of FP3 in combination with cetuximab on patient-derived tumor tissue xenograft models of primary colon carcinoma and related lymphatic and hepatic metastases

FP3 is an engineered protein which contains the extracellular domain 2 of vascular endothelial growth factor (VEGF) receptor 1 (Flt-1) and the extracellular domain 3 and 4 of VEGF receptor 2 (Flk-1, KDR) fused to the Fc portion of human immunoglobulin G1. Previous studies have demonstrated its antiangiogenic effects in vitro and in vivo, and its antitumor activity in vivo. Cetuximab is a monoclonal antibody against epidermal growth factor (EGF) receptor. Combined inhibition of VEGF and EGF signaling may act additively or synergistically. In this study, patient-derived tumor tissue (PDTT) xenograft models of primary colon carcinoma and lymphatic and hepatic metastases were established for assessment of the antitumor activity of FP3 in combination with cetuximab. Xenografts were treated with FP3 and cetuximab, alone or in combination. After tumor growth was confirmed, volume and microvessel density in tumors were evaluated. Levels of VEGF, EGFR and PCNA in the tumor were examined by immunohistochemical staining, and levels of related cell signaling pathway proteins were examined by western blotting. FP3 in combination with cetuximab showed significant antitumor activity in three xenograft models (primary colon carcinoma, lymphatic metastasis and hepatic metastasis). The microvessel density in tumor tissues treated with FP3 in combination with cetuximab was lower compared to that of the control. Antitumor activity of FP3 in combination with cetuximab was significantly higher than that of each agent alone in two xenograft models (colon carcinoma lymphatic metastasis and hepatic metastasis). This study indicated that addition of FP3 to cetuximab significantly improved tumor growth inhibition in the PDTT xenograft models of colon carcinoma lymphatic and hepatic metastases. Combination anti-VEGF (FP3) and anti-EGFR (cetuximab) therapies may represent a novel therapeutic strategy for the management of metastatic colon carcinoma.

The Tyrosine Kinase c-Src Directly Mediates Growth Factor-Induced Notch-1 and Furin Interaction and Notch-1 Activation in Pancreatic Cancer Cells

The proteolytic activity of Furin responsible for processing full length Notch-1 (p300) plays a critical role in Notch signaling. The amplitude and duration of Notch activity can be regulated at various points in the pathway, but there has been no report regarding regulation of the Notch-1-Furin interaction, despite its importance. In the present study, we found that the Notch-1-Furin interaction is regulated by the non-receptor tyrosine kinase, c-Src. c-Src and Notch-1 are physically associated, and this association is responsible for Notch-1 processing and activation. We also found that growth factor TGF-α, an EGFR ligand, and PDGF-BB, a PDGFR ligand, induce the Notch-1-Furin interaction mediated by c-Src. Our results support three new and provocative conclusions: (1) The association between Notch-1 and Furin is a well-regulated process; (2) Extracellular growth factor signals regulate this interaction, which is mediated by c-Src; (3) There is cross-talk between the plasma growth factor receptor-c-Src and Notch pathways. Co-localization of Notch-1 and c-Src was confirmed in xenograft tumor tissues and in the tissues of pancreatic cancer patients. Our findings have implications for the mechanism by which the Notch and growth factor receptor-c-Src signaling pathways regulate carcinogenesis and cancer cell growth.

Profiling Eph receptor expression in cells and tissues

The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.

Effects of cold ischemia and inflammatory tumor microenvironment on detection of PI3K/AKT and MAPK pathway activation patterns in clinical cancer samples

The accuracy of common markers for PI3K/AKT and MAPK pathway activation in preclinical and clinical cancer biomarker studies depends on phosphoepitope stability and changes of phosphorylation under ischemia. Herein, we define conditions under which phosphoepitope-specific duplex immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded tumor tissues reflects pathway activation in situ as accurately as possible, and identify activation patterns linked to mutational status, pathway dependency and tumor microenvironment in clinical tumor samples, cell culture and xenograft tissues. Systematically assessing robustness of pAKT, pERK1/2, pMEK1/2 and pmTOR detection and related markers in xenograft tissues exposed to ischemia, we show that control of preprocessing and ischemia times allows accurate interpretation of staining results. Phosphorylation patterns were then analyzed in 33 xenograft models and in 58 cases with breast cancer, including 21 paired samples of core-needle biopsies with corresponding mastectomy specimens, and 37 mastectomy samples obtained under rigorously controlled conditions minimizing ischemia time. Patterns of pAKT and pERK1/2 staining (predominant PI3K/AKT, predominant MAPK and concomitant activation) were associated with sensitivity to pathway inhibition and partially with the mutational status in cell lines and corresponding xenograft tumors. In contrast, no clear correlation between mutational status and staining patterns was observed in clinical breast cancer samples, suggesting that interaction with the human tumor microenvironment may interfere with the use of phosphoepitope-specific IHC as potential markers for pathway dependency. In contrast to core needle biopsies, surgically resected breast cancer samples showed evidence of severe signal changes comparable to those effects observed in xenograft tumors exposed to controlled ischemia.

Antitumor effect of FP3 in a patient-derived tumor tissue xenograft model of gastric carcinoma through an antiangiogenic mechanism

FP3 (KH902/KH903) is a novel vascular endothelial growth factor (VEGF) blocker with antiangiogenic properties. Previous studies revealed that FP3, a humanized fusion protein that combines ligand binding elements from the extracellular domains of VEGF receptors 1 and 2 and the Fc portion of IgG1, has an inhibitory effect on the VEGF-mediated proliferation and migration of human umbilical vein endothelial cells, and VEGF-mediated vessel sprouting of rat aortic rings in vitro. Thus, FP3 was considered as a new promising agent in treating human choroidal neovascularization (CNV) caused by age-related macular degeneration (AMD). FP3 also has an antitumor effect in a non-small cell lung cancer cell line (A549) xenograft model in nude mice. However, little is known of the direct effects of FP3 on tumor vessels. In this study, we investigated the effects of FP3 on blood vessels in a patient-derived tumor tissue (PDTT) xenograft model of gastric carcinoma, using large tumors with established vasculature. Treatment with FP3 caused robust and early changes in endothelial cells and pericytes of vessels in the PDTT xenograft model. Vascular density decreased and vascular sprouting was suppressed by treatment with FP3. Pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels achieved a more normal phenotype. Our results revealed that FP3 has a direct and rapid antiangiogenic effect on tumor vessels, which was achieved mainly via regression of tumor vasculature, inhibition of new and recurrent vessel growth, and normalization of existing tumor vasculature.

Development of an Immunohistochemical Protein Quantification System in Conjunction with Tissue Microarray Technology for Identifying Predictive Biomarkers for Phosphatidylinositol 3-Kinase Inhibitors

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers by gain-of-function mutations of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) or dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Therefore PI3K is thought to be a promising target for cancer therapy. Many agents targeting PI3K have been developed and some of them have been evaluated in clinical trials. In recent years, development of predictive biomarkers as companion diagnostics for molecular targeted drugs has become an important requirement for clinical development; however, no clinically established biomarkers that predict the efficacy of PI3K inhibitors have been found. We previously reported that expression of phosphorylated Akt determined by immunoblot analysis correlated with the antitumor efficacy of a PI3K inhibitor ZSTK474 in vitro and in vivo, suggesting that it might be used as a predictive biomarker. In this study, to evaluate biomarker candidates in in vivo tumor samples, we developed an immunohistochemical protein detection/quantification system in conjunction with the tissue microarray technology using a panel of 24 human tumor xenografts (JFCR24). We have clearly demonstrated that expression levels of phosphorylated v-akt murine thymoma viral oncogene homolog (Akt) and mitogen-activated protein kinase (MAPK) determined by this system significantly correlated with those determined by immunoblot analysis. As expected, PTEN status correlated with expression of phosphorylated Akt but not MAPK. Finally, we confirmed that phosphorylated Akt levels determined using this system correlated with the in vivo efficacy of ZSTK474. The present results indicate that the immunohistochemical protein detection/quantification system could be used to quantify expression of biomarker proteins in xenografted tumor tissues as well as in human tumor specimens to predict drug efficacy in future clinical trials.

Anti-tumor Activity of the Novel Hexahydrocannabinol Analog LYR-8 in Human Colorectal Tumor Xenograft Is Mediated through the Inhibition of Akt and Hypoxia-Inducible Factor-1α Activation

Cannabinoid compounds have been shown to exert anti-tumor effects by affecting angiogenesis, invasion, and metastasis. In the present study, we examined the action mechanism by which LYR-8, a novel hexahydrocannabinol analog, exerts anti-angiogenic and anti-tumor activity in human cancer xenografts. In the xenografted tumor tissues, LYR-8 significantly reduced the expression of hypoxia-inducible factor-1 alpha (HIF-1α), a transcription factor responsible for induction of angiogenesis-promoting factors, and its target genes, vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2). In HT-29 human colon cancer cells treated with a hypoxia-inducing agent (CoCl(2)), LYR-8 dose-dependently suppressed the induction of HIF-1α and subsequently its targets, VEGF and COX-2. In addition, highly elevated prostaglandin E(2) (PGE(2)) concentrations in CoCl(2)-treated HT-29 cells were also significantly suppressed by LYR-8. However, LYR-8 alone in the absence of CoCl(2) did not alter the basal expression of VEGF and COX-2, or PGE(2) production. Furthermore, LYR-8 effectively suppressed Akt signaling, which corresponded to the suppression of CoCl(2)-induced HIF-1α accumulation. Taken together, LYR-8 exerts anti-tumor effects through the inhibition of Akt and HIF-1α activation, and subsequently suppressing factors regulating tumor microenvironment, such as VEGF and COX-2. These results indicate a novel function of cannabinoid-like compound LYR-8 as an anti-tumor agent with a HIF-1α inhibitory activity.

OT1-03-03: Effect of Tamoxifen Therapy on Inhibition of Tumor Suppressor p53 by Estrogen Receptor.

Abstract Background: A large number of patients with estrogen receptor (ER)-positive tumors are resistant to tamoxifen (TAM). Although several plausible reasons for such resistance have been suggested, the mechanisms remain unclear. ER mediates effects of estrogen by promoting proliferation of breast cancer cells. Tumor suppressor protein p53 guards against tumorigenesis by preventing proliferation of cells with genomic damage. Dr. Das's laboratory previously reported that ER binds and functionally suppresses wild type p53 in human breast cancer cells and xenograft tumor tissue, and TAM is capable of inhibiting this interaction. We hypothesize that relieving suppression of wild type p53 by ER could be an important mechanism underlying TAM action in breast cancer. To test this hypothesis, we have initiated a pilot randomized clinical trial of 50 women with newly diagnosed ERa-positive breast cancer. Specific Aims: 1) To investigate the effect of a short pre-surgical intervention with TAM on the ER-p53 interaction in ER-positive, p53 wild type breast tumors and 2) To confirm the wild type status of p53 and analyze the functional status of the p53 pathway by monitoring expression of selected p53 target genes in tumors that have and have not been treated with TAM. Trial design: A randomized clinical trial in the pre-surgical setting was proposed with either TAM 20mg for four weeks vs. no intervention. Patients randomized to the TAM arm will undergo multiple pharmacokinetic and phamacodynamic measurements of TAM metabolites and genotyping for common polymorphisms of TAM metabolism genes, CYP2D6 and CYP3A4/5. Fresh tumor tissue will be harvested from all patients at the time of surgery for analysis of ER-p53 protein interaction using tissue chromatin immunoprecipitation (tissue ChIP) assay. p53 gene status will be determined by sequencing. RNA and protein expression of ER, p53, and a selected group of ER and p53 target genes in the diagnostic core biopsy and surgical specimens will be analyzed by quantitative real-time PCR (qRTPCR) and immunohistochemistry (IHC). TAM metabolites will also be measured in the tumor and the surrounding benign tissue. Eligibility criteria: Women greater or equal to eighteen years of age diagnosed with ER-positive invasive breast cancer (approximately 1cm in size) who will undergo primary surgical excision for their initial therapy are eligible. Women must not be pregnant, be on current hormonal therapy, or have a history of hypercoagulable syndrome or prior arterial or venous thrombosis. Statistical methods: Descriptive statistics such as frequencies and relative frequencies will be computed for all categorical variables. Numeric variables will be summarized using simple descriptive statistics such as mean, standard deviation, and quartiles. Ninety-five percent confidence intervals will be computed. ChIP data will be subjected to Fisher's Exact Test. IHC date will be analyzed by Wilcoxon-Mann-Whitney test. Present accrual and target accrual: We have accrued 17 patients to date and plan to accrue a total of 50 patients, 25 in each arm. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT1-03-03.