bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A44: Predictive biomarkers of efficacy to the hedgehog pathway inhibitor, GDC-0449, in advanced basal cell carcinoma and medulloblastoma in phase I studies
Authors: Yauch Robert , Januario Thomas , Fu Ling , Holcomb Thomas , Stinson Jeremy , Pujara Kana , Callahan Christopher , Koeppen Hartmut , Reddy Josina , Von Hoff Daniel , LoRusso Patricia , Rudin Charles , de Sauvage Frederic , Low Jennifer
Published: June 2012

The Hedgehog (Hh) signaling pathway has been directly implicated in the pathogenesis of basal cell carcinoma (BCC) and medulloblastoma (MB). Constitutive Hh signaling, most often due to underlying loss of function mutations in the inhibitory receptor Patched1 (PTCH1), has been demonstrated in a majority of common localized BCCs and approximately 25–30% of sporadic MB cases. In this study, we examined molecular correlatives of clinical efficacy in locally-advanced / metastatic BCC and MB patients treated with GDC-0449, an inhibitor of the Hh pathway that targets the serpentine receptor Smoothened (SMO). An analysis of Hh pathway transcriptional target gene expression in archival tissue biopsies provided evidence for active Hh signaling in patients exhibiting clinical benefit (objective response or disease stabilization) on therapy. This was further supported by the identification of either loss of function or activating mutations in PTCH1 or SMO, respectively. We were unable to provide evidence for active Hh signaling in 2 patients with progressive disease as best response, potentially underlying the reason for lack of efficacy in these patients. In addition to providing molecular support for Hh pathway activity in patients exhibiting treatment benefit, this is the first evidence for Hh pathway dysregulation in the previously uncharacterized, advanced BCC setting. Furthermore, the results begin to support the utility of transcriptional-based tests as a strategy to potentially select the subset of MB patients with Hh-driven disease that would likely benefit on GDC-0449.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A44.
Abstract A49: FDG-PET as an imaging biomarker for the response of xenograft tumors to IGF-1R inhibition
Authors: Wang Yan , Wang Yaolin , Wang Xiaoying , Hailey Judith , Lipari Philip , Seidel-Dugan Cindy , Larsson Steve
Published: June 2012

Insulin-like growth factor 1 receptor (IGF-1R) plays an important role in tumor cell growth and survival. Upon ligand stimulation, IGF-1R, a tyrosine kinase, phosphorylates tyrosine residues on two major substrates, IRS-1 and Shc, which subsequently signal through the Ras/MAPK and PI3 kinase/AKT pathways. Several anti-IGF-1R monoclonal antibodies are in early phase of clinical development for multiple cancer indications. Biomarker strategy is one of the key elements for targeted therapy development. Several avenues of biomarker assessments have been explored in preclinical models. Many of them are not practical in clinical settings due to lack of adequate tumor tissues accessible for biopsy. Positron emission tomography (PET) allows non-invasive and quantitative studies of biological processes in the tumor tissues. [18F]-fluorodeoxy-glucose (FDG) PET, especially, has become a technology that is available in increasing numbers of cancer centers. Here, we describe impact of a fully human anti-IGF-1R monoclonal antibody SCH 717454 on tumor proliferation index and metabolism in xenograft models. SCH 717454 inhibited the Ki67 marker expression and reduced the uptake of 18F-glucose uptake at much earlier time points than changes in tumor volumes. These data provide preclinical support for using FDG-PET as an early non-invasive method in assessing tumor response in clinical development of this agent.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A49.
Abstract A53: Tumor suppressor microRNA miR-34 inhibits human pancreatic and gastric cancer stem cells
Authors: Xu Liang , Ji Qing , Hao Xinbao , Tang Wenhua , Li Ling , DeSano Jeffrey T. , Fearon Eric R. , Lawrence Theodore S.
Published: June 2012

MicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. Among the target proteins regulated by miR-34 are Notch and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells.

We examined the roles of miR-34 in p53-mutant human pancreatic and gastric cancer cell lines and the potential link to cancer stem cells. Restoration of miR-34 expression in the cancer cells by miR-34 mimics or lentiviral miR-34 downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cancer cells to chemotherapy and radiation. We identified that CD44+/CD133+ cancer cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to a significant reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo. Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic and gastric cancer cells. Our data support that miR-34 may be involved in cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in cancer stem cell self-renewal and/or cell fate determination. Currently, we are exploring the nanoparticle-targeted delivery of miR-34 to cancer stem cells as a novel molecular therapy for human pancreatic/gastric cancer, based on our patented nanobiotechnology which has been approved by FDA for clinical trials.

Taken together, our results demonstrate that restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic/gastric cancer with loss of p53-miR34, potentially via inhibiting cancer stem cells. The project is funded by NIH grants CA121830, CA128220 and CA134655 (to L. Xu.).

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A53.
Abstract A54: STAT3 as a novel therapeutic target in human breast cancer stem cells
Authors: Lin Li , Hutzen Brian , Peng Zhengang , Lin Huey-Jen , Li Pui-Kai , Li Chenglong , Wicha Max , Lin Jiayuh
Published: June 2012

The American Cancer Society estimates that over 200,000 new cases of invasive breast cancer and over 40,000 deaths are expected among women in United States each year. Despite advances in chemotherapy and hormonal treatment, at the present time, metastatic breast cancer remains an incurable disease. Although a large number of chemotherapeutic agents have been developed that are able to cause the regression of metastatic breast cancers, these tumors almost always recur following chemotherapy treatment. It is possible that breast cancer stem cells or breast cancer initiating cells are responsible for the drug-resistance, metastasis, and recurrence of cancer. Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in many types of cancers, including breast cancer, and has emerged as an attractive molecular target for cancer treatment. However, whether or not STAT3 is activated in breast cancer stem cells and what role STAT3 signaling plays in cancer stem cells is still unknown. If STAT3 is activated in breast cancer stem cells, targeting STAT3 may offer a promising opportunity to eliminate breast cancer stem cells and prevent the recurrence of cancer. We examined STAT3 activation in breast cancer stem cells, which are characterized by an aldehyde dehydrogenase (ALDH)-positive subpopulation. Interestingly, we observed that the ALDH-positive subpopulation of breast cancer cells expressed higher levels of phosphorylated STAT3, an active form of STAT3, as compared to the ALDH-negative subpopulation and un-separated breast cancer cells, suggesting that STAT3 is activated in breast cancer stem cells. We also demonstrated that a novel STAT3 inhibitor, LLL12, inhibited STAT3 phosphorylation, cell viability, STAT3 downstream target gene expression, and induced apoptosis in breast cancer stem cells. In addition, the STAT3 inhibitors Stattic and LLL12 reduced ALDH-positive subpopulation and inhibited their tumorsphere forming capacity. Furthermore, in our pilot study in a mouse tumor model using breast cancer stem cells, we demonstrated that LLL12 can suppress tumor growth in vivo. This is the first report to demonstrate that persistent STAT3 phosphorylation is expressed in breast cancer stem cells and that this subpopulation of breast cancer stem cells is sensitive to STAT3 inhibitors. Our results suggest that STAT3 is a novel therapeutic target in breast cancer stem cells and inhibition of activated STAT3 in cancer stem cells may offer a more effective treatment for breast cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A54.
Abstract A7: Targeting the tumor microenvironment via inhibition of VEGF and PDGF to improve liposomal drug delivery in human non-small cell lung cancer xenografts
Authors: Tailor Tina D. , Hanna Gabi , Yarmolenko Pavel S. , Dewhirst Mark W.
Published: June 2012

Background: Pathological angiogenesis creates an abnormal microenvironment in tumors, characterized by elevated interstitial fluid pressure (IFP) and hypoxia. Judicious attenuation of angiogenic signals may “normalize” the vascular bed, making it more suitable for drug delivery and radiotherapy. In this work, we investigate the role of pazopanib, a small-molecule inhibitor of vascular endothelial growth factor (VEGF) receptors and platelet-derived growth factor (PDGF) receptors, on normalization parameters of IFP, vascular pericyte coverage, hypoxia, and liposomal drug delivery.

Methods: Nude mice bearing A549 human non-small cell lung cancer (NSCLC) xenografts were treated with pazopanib 100mg/kg (n=20) or control (n=20) for 8 days, followed by a single intravenous dose of Doxil (liposomal doxorubicin) 10mg/kg. 24 hours after Doxil delivery, tumor IFP was measured, and tumors were snap frozen and sectioned. In a second study, designed similarly to the previous (n=4 pazopanib 100mg/kg, n=4 control), EF5 (hypoxia marker) was delivered 3 hours prior to tumor harvest. Tissue sections were stained for vasculature and pericytes using anti-CD-31 (endothelial cell marker) and anti-alpha-smooth muscle actin, respectively. Hypoxia was assessed via immunostaining for CA-IX and EF5. High performance liquid chromatography was used to determine tumor doxorubicin concentration ([dox]), and the penetration distance of Doxil from CD-31 stained vessels was calculated using a MATLAB algorithm.

Results: Pazopanib treatment resulted in reduction of tumor IFP (116.571 ± 15.174 mmHg control vs. 61.000 ± 7.367 mmHg pazopanib, P = 0.0145) and decreased vessel density (251.228 ± 18.770 vessels/mm2 control vs. 101.313 ± 11.005 vessels/mm2 pazopanib, P < 0.0001). No differences in tumor [dox] were observed between groups (10.849 ± 1.785 ng/kg control vs. 12.274 ± 1.013 ng/mg pazopanib, P = 0.5956), however Doxil penetration improved in pazopanib-treated tumors (18.980 ± 0.687 m control vs. 32.886 ± 1.741 m pazopanib, P = 0.0006). Despite these trends towards normalization, tumor hypoxia, evaluated by CA-IX and EF5 expression, was increased in the pazopanib group (% CA-IX staining, 7.034 ± 0.988% control vs. 16.720 ± 1.728% pazopanib, P = 0.0005;% EF5 staining, 15.992 ± 2.610% control vs. 36.554 ± 4.617% pazopanib, P = 0.0074). Additionally, the proportion of pericyte-coated vessels was decreased in the pazopanib group (0.840 ± 0.0362 control vs. 0.675 ± 0.0495 pazopanib, P = 0.0160).

Conclusions: Our results suggest a role for VEGF/PDGF inhibition in modulating functional parameters of the tumor microenvironment and improving drug distribution. However, these changes may be mediated by stromal changes in response to PDGF-receptor blockade, rather than normalization.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A7.
Abstract A75: Dual inhibition of EGFR and ErbB2 during androgen ablation prevent the progression of prostate cancer cells to androgen independence
Authors: Chen Liqun , Madhav Anisha , Ghosh Paramita M.
Published: June 2012

Introduction: Patients with metastatic prostate cancer are treated with androgen ablation therapy which is initially effective, but most patients on this therapy ultimately become refractory to this treatment. Studies show that androgen withdrawal does not result in cell death, but simply cell growth arrest, whereas the development of androgen independence allows them to come out of the growth arrest and undergo cell cycle progression. Hence, induction of apoptosis during androgen ablation will prevent progression to androgen independence. The goal of the present project is to determine whether dual inhibition of EGFR and ErbB2 in prostate cancer cells during androgen withdrawal prevent their progression to androgen independence. EGFR, ErbB2, ErbB3 and ErbB4 constitute a family of receptor tyrosine kinases that require dimerization for complete activation.

Experimental Procedures: Androgen dependent LNCaP prostate cancer cells and its androgen-independent subline LNCaP-AI were treated with combinations of the EGFR inhibitors AG1478 or Erlotinib (Tarceva), and the ErbB2 inhibitors AG879 or Trastuzumab (Herceptin), either in the presence of androgens, or together with androgen withdrawal. These experiments were also conducteds in androgen receptor (AR)-null pRNS-1-1 cells transfected with vector only, wild type AR or mutant AR(T877A).

Results: Dual inhibition of EGFR and ErbB2 was more effective in inhibiting cell growth compared to single inhibition of either kinase. This effect was more pronounced in the absence of androgens compared to its presence, and was also more effective in androgen-dependent than in androgen-independent cells. Investigation of the mechanism of this effect demonstrated that dual inhibition of EGFR and ErbB2 also inhibited ErbB3 phosphorylation, likely by preventing its dimerization. Androgen withdrawal results in increased Akt phosphorylation, and we show here that dual EGFR/ErbB2 inhibition, but not single inhibition, prevented this effect and induced apoptosis. This effect is most pronounced in androgen sensitive cells undergoing androgen ablation, whereas in androgen-independent cells where Akt phosphorylation is already increased, the effect of the dual inhibition was less pronounced.

Conclusions: Our results indicate that cell survival during androgen withdrawal caused by an increase in Akt phosphorylation may be prevented by dual use of EGFR and ErbB2 inhibitors. If the cells can be killed during androgen ablation, then no cells will remain to progress to androgen independence. Hence, the effect of the dual inhibition prevents the development of androgen-independence in prostate cancer cells. The significance of these results is that they suggest that the use of dual EGFR/ErbB2 inhibitors during androgen ablation may prevent the development of androgen independence in patients undergoing androgen ablation therapy.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A75.
Abstract A84: Role of Akt pathway in the response of cetuximab in a resistant head and neck cell line
Authors: Rebucci Magali , Peixoto Paul , De Witte Amelie , Dewas Claire , Lartigau Eric , Lansiaux Amelie
Published: June 2012

The Epidermal Growth Factor Receptor (EGFR) is a central regulator of cell proliferation and differentiation in human cancers and is frequently overexpressed in many types of tumors. Cetuximab is a monoclonal antibody designed to block the EGFR activation, to induce the internalization of the receptor and to reduce the signaling pathway. However many tumors exert resistance to EGFR inhibitors. Accumulating evidence indicates that the response of cancer cells to cetuximab is a complex process that can be affected by multiple intrinsic and extrinsic resistance mechanisms. The aim of this study was to investigate the mechanisms of resistance to cetuximab treatment in vitro. We investigated the EGFR pathway and the receptor internalization after cetuximab treatment, alone or combined, and we compared results in CAL33 (HNSCC) and A431 (epidermoïd cancer) cell lines which overexpressed EGFR and present different sensitivities to cetuximab and to EGF (EGFR natural ligand).

Cetuximab induced a high growth inhibition and a high inhibition of ERK and AKT phosphorylation in A431. By contrast, cetuximab induced a lower growth inhibition, an ERK phosphorylation inhibition but any inhibition of the AKT pathway. The cetuximab sensitivity of these cell lines was thus different and the difference would be based on the AKT pathway. To verify this hypothesis, we used an EGFR tyrosine kinase inhibitor and an AKT inhibitor. Results have shown that the AKT pathway can be inhibited in CAL33 cell line by an EGFR tyrosine kinase inhibitor and an AKT inhibitor. While the cetuximab induced a strong growth inhibition in A431, this growth inhibition was weakly increased by the combination. In contrast, cetuximab induced a weak growth inhibition in CAL33 while the growth inhibition was much stronger with the combination than in A431. According to the central role of the EGFR internalization in the AKT pathway activation, we investigated the EGFR internalization role in these models. Preliminary results showed significant differences between these two cell lines. In conclusion, this study had shown some explanations to the limited efficacy to cetuximab in CAL33. Firstly, we have shown a persistent activation of AKT in CAL33 which might prevent the antitumor effect of cetuximab. Secondly, we have shown that the EGFR internalization and signal transduction mediation were different in CAL33 and A431 and this difference might contribute to the response to cetuximab. The AKT pathway appears as a central element in the cetuximab sensitivity in these models and the combination of cetuximab with an AKT inhibitor could be a good therapeutic option in HNSCC

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A84.
Abstract A87: Synthesis and structure-activity relationships of a series of epipodophyllotoxin polyamine conjugated derivatives vectorized for active polyamine transporter system in tumor cells, leading to the selection of F14512 for clinical trials
Authors: Imbert Thierry , Guminski Yves , Cugnasse Sandrine , Grousseaud Martial , Barret Jean-Marc , Kruczynski Anna , Vispe Stéphane , Gras Stéphane , Annereau Jean-Philippe , Vandenberghe Isabelle , Stennevin Aline , Lacastaignaratte Laurence , Novosad Natacha , Pourtau Sandrine , Roy Sabine , Castano Caroline , Blanchet Jean-Christophe , Guilbaud Nicolas , Bailly Christian
Published: June 2012

One of the major concerns for chemotherapy is the selective targeting of drugs into highly proliferative cancer cells. Natural polyamines (spermine, spermidine, putrescine) are essential for the regulation of cellular growth and differentiation. Due to their highly proliferative nature, cancer cells have a pronounced need to import polyamines from their external environment, through the polyamine transporter system (PTS). On the basis of this biological mechanism, we vectorized the new cytotoxic anticancer compound F14512, a epipodophyllotoxin — spermine conjugate, into tumor cells.1 Here we present the synthesis and the structure-activity relationships of a new series of compounds constituted by an podophyllotoxin core tethered with a polyamine moiety with a variable spacer. Two synthetic strategies with protected polyamines, and a direct 3 steps synthesis of F14512 from natural podophyllotoxin and spermine without any protection are presented.2 This series of topoisomerase II inhibitors were checked for their cytotoxicity on A549 lung cancer cell line, displaying marked potency up to nM range. Cancer cell internalization through PTS was assessed by selective cytotoxicity on different PTS expressing cell lines, and by competition experiments. Our results displayed a potent specificity for the conjugated tetramine (spermine) compounds, which were more recognized than the triamine (spermidine) ones, while mono and diamines showed no selectivity. Lead compounds were also tested in vivo and proved potent antitumor activity. This series of new water-soluble cytotoxic compounds culminates with the selection of F14512 for clinical trials.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A87.
Abstract B103: Integrative genomic and proteomic analyses identify novel targets for Lkb1 deficient metastatic lung tumors
Authors: Carretero Julian , Shimamura Takeshi , Rikova Klarisa , Gu Ting-Lei , Borgman Christa L. , Buttarazzi S. , McNamara Kate L. , Brandstetter Kathleyn A. , Silva Jeffrey C. , Shapiro Geoffrey I. , Castrillon Diego H. , Maira Michel , García-Echeverría Carlos , Kim Carla F. , Bardeesy Nabeel , Sharpless Norman E. , Kim William Y. , Engelman Jeffrey A. , Wong Kwok K.
Published: June 2012

Lung cancer is the leading cause of cancer related mortality worldwide. Gene expression profiling of primary human lung tumors has successfully led to the identification of aberrant signalling pathways involved in the initiation of non-small cell lung cancer (NSCLC). However, advances in defining the pathways deregulated in lung cancer progression and metastases have been limited. The process of tumor metastasis is a complex, multistage process that involves both metastasis initiation functions, such as invasion and angiogenesis, as well as metastasis progression functions such as extravasation and survival. In mice, lung-specific deletion of the Lkb1 tumor suppressor gene and activation of mutant Kras results in lung tumors with a high penetrance of lymph node and distant metastases. However, which of the several candidate pathways are responsible for the pro-metastatic effects of Lkb1 loss has yet to be defined. To identify altered signal transduction pathways involved in the progression and metastases of Lkb1 deficient lung tumors we have integrated genomic and phosphoproteomic signatures of primary and metastastic, Lkb1 deficient lung tumors to identify differentially activated signal transduction pathways. These studies revealed a number of signatures associated with the process of metastasis such as EMT, stem cell phenotype, growth factor signaling and focal adhesion. Moreover, several tyrosine kinases, including the non-receptor tyrosine kinase Src and the focal adhesion kinase, are activated in Lkb1 deficient primary and metastatic lung tumors, resulting in increased motility and Src-dependent adhesion of LKB1 deficient cells. Collectively, these results point towards a mechanism behind the increased propensity for metastases seen in Lkb1 deficient lung tumors and identify molecularly targetable pathways for the treatment of LKB1 deficient NSCLC in humans.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B103.
Abstract B109: Modulation of tumor-associated lymphatic function by the VEGF-C axis
Authors: Caunt Maresa , Gogineni Alvin , Weimer Robby , Bagri Anil
Published: June 2012

Lymph vasculature associated with solid tumors provides an exit route for disseminating metastatic cells. VEGF-C, a member of the vascular endothelial growth factor family, is upregulated in a range of metastatic tumor types. VEGF-C is known to mediate lymphatic development and is implicated in modulating lymph vessel function. These observations have lead to the hypothesis that VEGF-C expression in tumors may promote metastatic cell dissemination by augmenting lymph morphology or function. To explore this hypothesis we have developed longitudinal assays based on near-infrared fluorescence imaging technologies to study lymphatics associated with primary tumors. In preliminary experiments we observe a ∼ 50% increase in bulk transport through lymph beds draining VEGF-C(+) primary tumors. This increase in transport correlates with a ∼50% increase in contractile events along the draining vessels, suggesting that chronic production of VEGF-C promotes lymph transport in part by stimulating contractility. We are currently testing agents which block VEGF-C-mediated signaling pathways to determine if reduced signaling correlates with reduced transport and rates of metastatic events.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B109.
Abstract B113: Role of I-TAC-binding receptors CXCR3 and CXCR7 in biology of various tumor cell lines
Authors: Miekus Katarzyna , Jarocha Danuta , Trzyna Elzbieta , Majka Marcin
Published: June 2012

Introduction: Chemokines and its receptors stimulate tumor growth, migration and invasion. SDF-1-CXCR4 axis plays a major role in these processes. However, other chemokines and their receptors are also important for tumor biology. Recently, SDF-1 was shown to bind with high affinity to the orphan receptor RDC1, later renamed CXCR7. In human tissues, CXCR7 expression has been described on endothelial cells and on some tumor cell lines. Recent studies confirmed a critical role for CXCR7/RDC1 in tumor vascular formation, angiogenesis and promotion of growth of breast and lung cancer in vivo.

Objectives: In this study we evaluated the expression and function of CXCR3 and CXCR7 receptors in cervical carcinoma, rhabdomyosarcoma and glioblastoma cell lines. The main goal of this study was to explore the role of CXCR7 in biology of rhabdomyosarcoma, cervical carcinoma and glioblastoma.

Material and Methods: Several cell lines were used in the study: rhabdomyosarcoma (SMS-CTR, RD, RH18, RH28, CW9019, RH30), cervical carcinoma (HeLa, HTB-34, HTB-35) and glioblastoma (LN18, LN229, T98G). To study the expression and function of CXCR3 and CXCR7 receptor real-time PCR, flow cytometry, western blot and chemotaxis assays were used.

Results: We tested 12 different cell lines and found that all of them expressed CXCR7 and CXCR3 at mRNA and protein level. We noticed higher expression of CXCR7 on more aggressive tumor cell lines (e.g. CXCR7 expression differed between rhabdomyosarcoma subtypes and it was highly expressed by alveolar rhabdomyosarcoma and expressed at the low level by embryonal rhabdomyosarcoma). Since I-TAC binds CXCR3 receptor as well, its expression was also examined by flow cytometry. We observed expression of CXCR3 receptor on all tested cell lines except HeLa and CW9019 and these cell lines were chosen to check functionality of the CXCR7 receptor. Upon I-TAC stimulation the phosphorylation of AKT and MAPK was observed. We did not observed the migration of tumor cells toward I-TAC gradient. However, at the low dose I-TAC sensitized tumor cells toward SDF-1 gradient and synergized with SDF-1 in activation of intracellular pathways.

Conclusions: We found that CXCR7 is expressed on various cell lines and its expression might correlate with aggressiveness of tumor. I-TAC, the ligand for CXCR7, was able to activate signaling transduction pathways responsible for tumor growth and survival.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B113.
Abstract B115: The epithelial to mesenchymal transition is impaired in colon cancer cells with microsatellite instability
Authors: Pino Maria S. , Kikuchi Hirotoshi , Zeng Min , Herraiz Maria-Teresa , Sperduti Isabella , Berger David , Park Do-Youn , Iafrate A. John , Zukerberg Lawrence R. , Chung Daniel C.
Published: June 2012

Colorectal cancers (CRC) displaying DNA microsatellite instability (MSI) are associated with a favorable natural history, but the molecular basis for this observation has not been defined. We sought to determine whether the epithelial-to-mesenchymal transition (EMT), a highly conserved process involved in embryogenesis as well as in tumor progression, invasion, and metastasis, is impaired in MSI-positive CRCs that characteristically have a mutant transforming growth factor-β receptor type II (TGFBR2) gene. The induction of EMT by TGF-β1 was analyzed by phase contrast microscopy, immunofluorescence, qRT-PCR, immunoblotting, and cellular migration and invasion assays in MSS (SW480 and HT29) and MSI (DLD1 and HCT116) colon cancer cell lines. Expression of epithelial (E-cadherin) and mesenchymal (vimentin and N-cadherin) markers was evaluated by immunohistochemistry and qRT-PCR in 129 human colorectal tumors. TGF-β1 induced changes in cellular morphology from an epithelial to a fibroblasticlike morphology, changes in gene and protein expression with a reduction in E-cadherin and induction of vimentin, and changes in motility and invasion consistent with the occurrence of EMT only in MSS colon cancer cells. These effects did not require Smad4 but depended upon the recruitment of ERK. Cells with MSI and mutant TGFBR2 failed to exhibit any of these changes in response to TGF-β1. However, tumor cells with MSI but wildtype TGFBR2 underwent EMT in response to TGF-β1, indicating that TGFBR2 genotype is a key determinant of the EMT response in tumors with MSI. In human colorectal tumors, expression of the EMT markers N-cadherin and vimentin was significantly associated with adverse clinicopathologic features and the absence of MSI. These findings define a unique genotype-phenotype relationship between TGFBR2 and EMT that may contribute to the improved prognosis consistently observed in colon cancers with MSI. Furthermore, these results suggest a potential rationale for the therapeutic inhibition of TGF-β signaling in MSS colorectal tumors.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B115.
Abstract B119: Insulin-like growth factor 1 receptor mutants are inhibited by figitumumab (CP-751, 871) in vitro
Authors: Reynolds Jennifer M. , Lloyd David B. , Bentivegna Steven C. , Seymour Albert B. , Borzillo Gary V. , Gualberto Antonio
Published: June 2012

To test the hypothesis that mutant insulin-like growth factor 1 receptor (IGF-1R) protein may differentially respond to inhibition by figitumumab (CP-751, 871), potentially necessitating patient screening, we studied the in vitro efficacy of figitumumab against seven identified non-synonymous somatic IGF-1R mutations. The IGF-1R signaling pathway has been implicated in numerous aspects of tumor biology, and inhibition of this pathway has been shown to provide beneficial anti-tumor effects. Figitumumab is a fully human monoclonal IgG2 antibody against IGF-1R that has been shown to block ligand binding, inhibit IGF-1R tyrosine kinase activity, and induce IGF-1R down-modulation. There have been reports in the literature of targeted cancer therapies being affected by somatic mutations within the target of the therapy itself as well as downstream in the signaling cascade. To identify somatic DNA mutations within the IGF1R gene, 46 colon and 46 lung matched normal/tumor sets were screened using a mismatch repair detection method. The screening process identified seven unique mutations in either colon or lung cancer samples that altered the IGFIR coding sequence; 5 in colon and 2 in lung. These mutations were distributed across various domains of the receptor with three located in the extracellular alpha chain and four in the intracellular beta chain. Of the four located in the intracellular beta chain, three were in the tyrosine kinase domain. To study the effects of these mutations on the kinase activity of the receptor and the ability to inhibit the mutant receptors with figitumumab, stable cell lines containing wild-type and the mutant receptors were created and evaluated by ELISA for down-regulation of the IGF-1R protein and IGF-1R phosphorylation. All seven mutant forms of the protein, as well as wild-type IGF-1R were inhibited by figitumumab. Six of the seven mutant proteins maintained dependency on ligand stimulation for kinase activity while one mutant, a glycine to arginine change at amino acid position 1199, lacked kinase activity all together. The results of our experiments suggest that figitumumab effectively inhibited the wild-type and mutant IGF-1R receptors in this study and therefore screening of figitumumab patients for these mutations should not be necessary.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B119.
Abstract B122: Human monoclonal antibodies to sialyl-Lewis-A with potent CDC and ADCC activity
Authors: Sawada Ritsuko , Sun Shu-Man , Hong Feng , Ragupathi Govind , Livingston Philip O. , Scholz Wolfgang W.
Published: June 2012

The carbohydrate antigen sialyl-Lewis A (sLea) is widely expressed on epithelial tumors of the gastrointestinal tract, on breast cancer cells, and also on small cell lung cancer cells, but is expressed minimally or not at all on normal tissues. sLea serves as a ligand for epithelial leukocyte adhesion molecules and higher expression of sLea was observed in patients with greater node involvement. Since over-expression of sLea appears to be a key event in invasion and metastasis of many tumor cells and tumor cells expressing sLea are highly susceptible to antibody mediated lysis mechanisms, sLea presents an attractive molecular target for tumor therapy in a minimal disease setting.

Here we report the discovery and initial characterization of fully human antibodies that were generated from blood lymphocytes from individuals immunized with sLea vaccine at MSKCC. Antibodies were identified by ELISA using sLea-HSA conjugates and binding to the native antigen was verified by FACS analysis on DMS-79 cells. Two antibodies were selected for further studies based on the apparent high affinity, which was estimated by Biacore at 0.14 nM for 5B1 (IgG/λ) and 0.04 nM for 7E3 (IgM/κ). These antibodies did not bind to sialyl-Lewis X, Lewis A and other related carbohydrates. Both antibodies have been expressed as fully functional human recombinant antibodies in CHO cells. Complement dependent cytotoxicity (CDC) against DMS-79 cells was approximately 60% and 70–90% for r5B1 and r7E3, respectively. Moreover, r5B1 antibodies showed approximately 50% ADCC of DMS-79 cells with human NK cells (at 5:1 ratio) and 80–90% ADCC with human peripheral blood mononuclear cells with two different blood donors (at 100:1 ratio). These results are very encouraging and we believe that further studies to scale up and test these antibodies in various tumor challenge models are warranted. Since sLea is widely expressed on human cancers, such antibodies could eventually find utility in approximately half of the new cancer cases occurring each year.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B122.
Abstract B126: Potent antigen-specific anti-tumor activity observed with antibody-drug conjugates (ADCs) made using a new class of DNA-crosslinking agents
Authors: Miller Michael , Fishkin Nathan , Li Wei , Leece Barbara , Mayo Michele , Jones Gregory , Reid Emily , Archer Katie , Maloney Erin , Kovtun Yelena , Pinkas Jan , Singh Rajeeva , Chari Ravi
Published: June 2012

The clinical support for ADC therapeutics has expanded as more highly-engineered ADCs advance in human clinical testing. Most of the ADCs now in clinical testing contain a tubulin-acting compound (a maytansine or dolastatin derivative) as the cytotoxic agent. While tubulin-acting agents can be effective against many different types of cancers, some cancers are more responsive to DNA-acting agents. To expand the therapeutic potential for ADCs, we sought to develop a new class of cytotoxic agents with a novel, DNA-acting mechanism of action for use with tumor-targeting antibodies. Herein, we report the development of our IGN family of cytotoxic agents. These IGN agents comprise indolino-benzodiazepine dimers that are highly potent by virtue of their ability to alkylate and crosslink DNA. This novel class of compounds demonstrated sequence-selective DNA adduct formation in vitro and cytotoxicity in the picomolar range towards cultured human cancer cells. The intense potency of these compounds, along with their desired aqueous solubility and stability, make them ideally suited for use in ADCs. A lead compound from this class was conjugated to an EpCAM-binding antibody, B38.1, and to a CD33-binding antibody, huMy9-6, through amide bonds. The B38.1-IGN conjugate was highly potent against three different EpCAM-expressing cell lines - COLO 205, LoVo and OVCAR-3 - with IC50 values of 1 pM, 5 pM and 18 pM, respectively. The addition of excess unconjugated B38.1 antibody abolished this cytotoxic effect, demonstrating that the activity of the conjugate is antigen specific. The B38.1-IGN conjugate was considerably less potent towards the antigen-negative Namalwa cell line, with an IC50 value of >1 nM, further demonstrating antigen specificity. Similar potent cytotoxicity was seen with a huMy9-6-IGN conjugate targeting the CD33-positive human promyelocytic leukemia cell line, NB4 (IC50 ∼4pM), in spite of the low antigen expression level (∼10,000 molecules/cell) in this cell line. Of particular interest, the B38.1-IGN conjugate also was potent towards multidrug resistant cancer cells. B38.1-IGN had a IC50 value of 14 pM for COLO 205MDR, a COLO 205 clone engineered to overexpress MDR1 transporter, and 7 pM for HCT-15, an EpCAM-expressing cell line that naturally expresses MDR1. Antibody-IGN conjugates demonstrated dose-dependent activity in multiple human tumor xenograft models in mice, with anti-tumor activity observed at non-toxic doses. The unique mechanism of action of the IGN class of compounds, and the high antigen-specific potency of antibody-IGN conjugates seen in vitro and in vivo, provides a promising new cytotoxic agent for use in the development of new ADCs.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B126.
Abstract B128: Affinity optimization of an anti-MUC1 antibody, (HuHMFG1) also enhances ADCC activity
Authors: Doran Ben , Ritchie Christy , Dhokia Babu , Rogers Paul , Jones David
Published: June 2012

HuHMFG1 (AS1402) is a humanized antibody which binds to MUC1, a cell surface glycoprotein which is over-expressed on tumor cells. A phase II clinical trial of huHMFG1 in metastatic breast cancer was recently terminated prior to completion as it was thought unlikely to give sufficiently positive efficacy findings.

HuHMFG1 contains a potential glycosylation site within the variable region; this is partially glycosylated in the drug product. The presence of the glycan reduces the affinity of the antibody for MUC1, and enzymatic deglycosylation of huHMFG1 increases affinity 3-fold. The asparagine residue which undergoes partial glycosylation (N56) was mutated to all other amino acids and binding affinities to antigen determined: several mutants exhibited enhanced binding to MUC1. The mutation to glycine (N56G) demonstrated approximately 10-fold increased affinity as assessed by antigen binding ELISA and cell binding ELISA against a variety of MUC1-expressing cell lines; BIAcore analysis further supported these observations.

The primary mechanism of action by which huHMFG1 is proposed to function is antibody dependent cell mediated cytotoxicity (ADCC). The N56G mutant exhibited 10 to 20-fold increased ADCC activity compared to huHMFG1 (5-10% of the huHMFG1 dose was required to elicit maximum cell kill); in addition the percentage of target cells killed also increased.

This second generation huHMFG1 exhibits increased antigen affinity and ADCC activity and furthermore has the potential to offer improved in vivo activity.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B128.
Abstract B129: Preclinical assessment of targeting insulin-like growth factor-2 in bone metastasis from prostate cancer by a human neutralizing antibody (m610) and human adult bone implanted model
Authors: Kimura Taichi , Kuwata Takeshi , Ashimine Satoshi , Nagai Kanji , Feng Yang , Dimitrov Dimiter S. , Saito Seiichi , Ochiai Atsushi
Published: June 2012

Background: Insulin-like growth factor (IGF)-2 activates IGF-1 receptor (IGF-1R) as well as insulin receptor (IR). Recently, it is becoming increasingly evident that IR activation by IGF-2 enhances the growth of neoplasms such as Ewing sarcoma and breast cancer in addition to the IGF-1R activation. Advanced prostate cancer frequently and organ-specifically metastasizes to the bone, where the IGF-2 is abundant. However, the role of IGF-2 in bone metastasis from prostate cancer is still uncertain. The present study was aimed at exploring the role of IGF-2 in the growth of prostate cancer cells in a human bone environment and the therapeutic potential of inhibiting IGF-2 by a neutralizing antibody in the bone metastasis.

Materials and Methods: Using our human neutralizing antibody to IGF-2 (m610) and human adult bone implanted mouse model, we investigated whether inhibiting IGF-2 suppresses the growth of prostate cancer cells in the human bone environment. Human MDA PCa 2b prostate cancer cells were inoculated into human adult bone implanted into mammary fat pad of NOD/SCID mice or inoculated into mammary fat pad of the mice without human bone implantation. The mice were treated with m610 or a control antibody once weekly for 4 weeks immediately after inoculation with MDA PCa 2b cells. At 4 weeks after the treatments, histological analyses of the obtained specimens were performed.

Results: M610 treatment significantly decreased the MDA PCa 2b tumor area in the human bone compared with the control. Ki-67 immunostaining revealed that the percentage of proliferating cancer cells in the m610-treated bone tumor sections was significantly lower than that in the control. M610 had no effect on MDA PCa 2b tumor growth in the absence of implanted human bone. IGF-1R and IR expressions on the MDA PCa 2b tumors were confirmed by immunostaining. M610 prevented in vitro IGF-2-induced cell proliferation, phosphorylation of the receptors and phosphorylation of Akt in MDA PCa 2b cells.

Conclusions: These results demonstrate that IGF-2 plays a crucial role in the prostate cancer cell growth in human bone. Targeting IGF-2 by neutralizing antibodies would be a promising therapeutic approach to preventing bone metastasis from prostate cancer.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B129.
Abstract B150: Toyocamycin inhibits ER stress-induced XBP1 mRNA splicing through IRE1α activation
Authors: Tashiro Etsu , Yokouchi Yumi , Yamamoto Kohta , Imoto Masaya
Published: June 2012

Poorly vascularized solid-tumor cells encounter a range of cytotoxic conditions such as hypoxia, nutrient deprivation and pH changes. These cytotoxic conditions in solid tumor are considered to cause ER stress. There is now abundant evidence to suggest that ER stress response is activated in various tumors to protect tumor cells from cytotoxic condition-induced apoptosis. XBP1 is considered to be a critical transcription factor of this process and required for tumor survival. Therefore, small molecule inhibitors of XBP1 activation would provide a new type of therapeutic strategy for cancer treatment. In this study, we screened small molecule inhibitors of ER stress-induced XBP1 activation from culture broth of microorganisms and we identified toyocamycin (H, Nishimura et al.: J. Antibiot., 9, 60 (1956)). Thus, we investigated the mechanism by which toyocamycin inhibited ER stress-induced XBP1 activation.

XBP1 is activated by IRE1α-mediated unconventional splicing of XBP1 mRNA, which resulted in creating a translational frame shift to produce an active XBP1 transcription factor. In HeLa cells, toyocamycin inhibited the transcriptional activity of XBP1, since toyocamycin suppressed tunicamycin or 2-deoxy-glucose, a well known ER stressor, -induced GRP78, EDEM, and ERdj4 expression that are known to be transcriptionaly regulated by active XBP1. Furthermore, toyocamycin inhibited ER stress-induced endogenous XBP1 mRNA splicing evaluated by RT-PCR. Although toyocamycin is known as RNA synthesis inhibitor, toyocamycin could not decrease the expression level of XBP1 mRNA. Thus, the inhibitory activity of toyocamycin against XBP1 activation is not due to the RNA synthesis inhibition. On the other hand, toyocamycin was unable to suppress IRE1α-mediated XBP1 mRNA cleavage in vitro. Therefore, toyocamycin is strongly suggested to inhibit ER stress-induced IRE1 activation.

Finally, we examined the effect of toyocamycin on cell viability under ER stress condition, because XBP1 inhibitor is considered to suppress ER stress-induced cytoprotective effect thereby inducing selective killing of stressed cells. Although toyocamycin had only a weak activity to induce cell death in HeLa cells, toyocamycin synergistically induced cell death when toyocamycin was incubated with 2-deoxy-glucose or tunicamycin. These results indicated that toyocamycin selectively induces cell death under ER stress condition. Taken together, our results suggested that XBP1 is a potent molecular target for developing anti-cancer drug.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B150.
Abstract B155: Effects of bamboo extract on breast cancer
Authors: Ton That Nam Tran S. , Samonte Sabrina-Lynn , Berry Marla J. , Panee Jun
Published: June 2012

Breast cancer is the most common cancer among women in the United States, and worldwide over one million new cases are diagnosed each year. Preliminary studies by Panee and colleagues demonstrated that 0.5% (w/w) dietary supplementation with an ethanol/water extract from bamboo Phyllostachys edulis inhibited the incidence of 7,12-dimethylbenz[a]anthracene (DMBA, a carcinogen)-induced mammary tumors in female Sprague-Dawley rats by 44% and dramatically reduced tumor multiplicity. This project has aimed to delineate the anti-breast cancer mechanisms of this bamboo extract (BEX) in the aforementioned rat model. We hypothesized that BEX may alter the gene expression of key proteins involved in estrogen- and/or growth factor-signaling pathways and xenobiotic biotransforming enzymes in the mammary tumors. To test this hypothesis, we extracted RNA from individual mammary tumors that were harvested from each rat. Then, we synthesized complementary DNA (cDNA) and combined the cDNA samples according to each rat's dietary group— BEX-supplemented rat diet or control rat diet. Using quantitative real-time polymerase chain reaction, we measured expression levels of target genes that included the following: estrogen receptors alpha and beta (ER-α and -β), human epidermal growth factor receptor 2 (HER2), and all glutathioine-s-transferase (GST) isoforms. Comparison between study groups revealed that BEX-supplementation significantly downregulated the gene expression of ER-α, ER-β, and HER2 in the mammary tumors by 65%, 70%, and 67% respectively (p < 0.0001). Additionally, the mammary tumors expressed all GST isoforms, whereby GSTa4 and GSTp1 resulted in high abundant values. Interestingly, BEX supplementation also significantly downregulated the gene expression of GSTa4 and GSTp1 by 32% and 31% respectively (p < 0.005). Our observations have indicated that the downregulation of ER-α, ER-β, and HER2 gene expression in the mammary tumors may contribute to the anti-breast cancer effect of BEX, and that BEX may also reduce chemoresistance in the mammary tumors by inhibiting the gene expression of xenobiotic biotransforming enzyme GST.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B155.