bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 723: GRB7 promotes doxorubicin resistance in triple-negative breast cancer cells via multiple signaling pathways
Authors: Lofgren Kristopher A. , Calvo Verónica , Giricz Orsi , Kenny Paraic A.
Published: August 2015

Triple-negative breast cancer (TNBC) lacks expression of the estrogen and progesterone steroid receptors and HER2; thus TNBC patients cannot benefit from the recent successes of targeted therapies, and instead rely on cytotoxic chemotherapeutic cocktails. As such, tumor recurrence is high and patient prognosis is poor.

GRB7 is a soluble adaptor protein required for cell migration, invasion, and proliferation in TNBC. We have previously reported that a high GRB7 mRNA level was the strongest predictor of tumor recurrence in a cohort of 246 TNBC patients treated with doxorubicin. GRB7 was confirmed in a smaller, independent cohort as an indicator of poor prognosis. After the observation of both decreased doxorubicin sensitivity and elevated GRB7 expression levels in TNBC tumors, we hypothesized that GRB7 was mediating a signaling pathway critical for drug resistance and cell survival.

The current study utilizes isogenic MDA-MB-231 breast cancer cell lines to examine the impact of altered GRB7 levels on TNBC cell signaling and chemosensitivity. Through stable overexpression and knock-down of GRB7, we have identified multiple downstream kinases with altered phosphorylation in basal growth conditions (Akt, Erk, and Jnk) that remain responsive to EGF stimulation. Treatment of TNBC cell lines with G7-18NATE, a specific peptide inhibitor of GRB7, sensitized TNBC cells to doxorubicin. Therefore, we created stable cell lines with constitutively active Akt and Map2k1 and the overexpression of MKK7 in order to determine whether the activation of these kinases individually have novel roles in GRB7-mediated chemoresistance and TNBC cell biology associated with the malignant phenotype, such as cell migration and invasion.

This study provides a strong rationale for targeting GRB7 or GRB7-dependent pathways in combination with cytotoxic chemotherapy for GRB7-high TNBC patients. Delineating the signaling events downstream of GRB7 in TNBC could provide further insight into improving the therapeutic index with combinatorial approaches.

Citation Format: Kristopher A. Lofgren, Verónica Calvo, Orsi Giricz, Paraic A. Kenny. GRB7 promotes doxorubicin resistance in triple-negative breast cancer cells via multiple signaling pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 723. doi:10.1158/1538-7445.AM2015-723
Abstract 725: Restoration of miR-100 sensitizes luminal A breast cancer to paclitaxel treatment by targeting mTOR
Authors: Zhang Baotong , Zhao Ranran , He Yuan , Fu Xing , Dong Jin-Tang
Published: August 2015

Based on molecular profiling, immunohistochemical detection of ERα, PR and Her2, and morphological evaluation, breast cancer is classified into five subtypes: Luminal A, Luminal B, Her2, Basal-like, and Normal-like. Each subtype has distinct prognosis and treatment response. The luminal A subtype nicely responds to endocrine therapy with a good prognosis, but is less responsive to chemotherapies including paclitaxel. According to a previous study, patients with luminal A breast cancer do not benefit from the addition of paclitaxel to their chemotherapies, but the underlying mechanisms are still not clear. We found that the expression of miRNA miR-100 is dramatically lower in luminal breast cancer cell lines compared to the basal-like subtype. In human breast cancer specimens, while miR-100 expression was downregulated when compared to the matched normal breast tissues, the downregulation in luminal A subtype tumors was more dramatic when compared to other subtypes. We therefore propose that lower levels of miR-100 expression could be responsible for decreased sensitivity of luminal A breast cancer to paclitaxel. To test this hypothesis, we expressed miR-100 in the MCF-7 luminal A breast cancer cell line, and found that expression of miR-100 sensitized MCF-7 cells to paclitaxel treatment by promoting paclitaxel-induced apoptosis and cell cycle arrest. We also suppressed miR-100 expression in the MDA-MB-231 basal-like cell line, and found that miR-100 downregulation desensitized MDA-MB-231 cells to paclitaxel. Tumorigenesis experiments in nude mice further support a role of miR-100 in luminal breast cancer's response to paclitaxel treatment. mTOR, an important player in the PI3K/mTOR signaling pathway, has been identified as a downstream target of miR-100, and combination of rapamycin, a mTOR inhibitor, with paclitaxel enhances the effect of chemotherapy in breast cancer. We found that miR-100 expression inhibited mTOR expression in MCF-7 cells, while suppression of miR-100 increased mTOR expression in MDA-MB-231 cells. These results suggest that miR-100 sensitizes luminal A breast cancer cells to paclitaxel via targeting mTOR. They also provide an explanation for the resistance of luminal A breast cancer to paclitaxel treatment.

Citation Format: Baotong Zhang, Ranran Zhao, Yuan He, Xing Fu, Jin-Tang Dong. Restoration of miR-100 sensitizes luminal A breast cancer to paclitaxel treatment by targeting mTOR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 725. doi:10.1158/1538-7445.AM2015-725
Abstract 726: Constitutive NF-kB-activation loop enhances resistance to trastuzumab in HER2 positive luminal B breast cancer
Authors: Kanzaki Hirotaka , Makhopadhya Nishit , Cui Xiaojiang , Ramanujan Krishnan V. , Takeda Tatsuaki , Kitamura Yoshihisa , Sendo Toshiaki , Murali Ramachandran
Published: August 2015

[Purpose]

Luminal B breast cancer patients are significantly at the risk of relapse with in 5 years. Acquired resistance to the receptor tyrosine kinase HER2 targeting antibody trastuzumab is a major clinical problem in the treatment of recurrent HER2-positive breast cancer. The molecular mechanisms leading to trastuzumab resistance (TZR) remain unclear in luminal B breast cancer. Here, we investigated the mechanism of TZR in HER2-positive luminal B breast cancer cells with acquired resistance.

[Methods and Results]

We generated trastuzumab resistant breast cancer cell line (BT-474-R) by treating BT-474 cells with trastuzumab for over long-term. Initial validation showed that HER2 overexpression in the BT-474-R cells remained unaltered suggesting that probably HER2 independent signaling pathway is involved in the development of TZR. We demonstrated that NF-κB is constitutively activated in the BT-474-R cells by quantitative realtime RT-PCR, western blotting and immunofluorescent microscopy. Expression of pro-inflammatory cytokines were also demonstrated. Pharmacologic inhibition of NF-κB improved sensitivity to trastuzumab in BT-474-R cells. We identified both IKK-α and IKK-β as the major determinants of the activation of the NF-κB signaling in the resistant cells.

[Conclusion]

Our study suggests by activating NF-κB pathway, luminal B cells may facilitate acquiring basal-like, Her2+ phenotype a notion consistent with “progression through grade” hypothesis. Finally, our study suggests that blocking NF-κB will provide a new treatment option to limit relapse in HER2 positive breast cancer patients treated with trastuzumab.

Citation Format: Hirotaka Kanzaki, Nishit Makhopadhya, Xiaojiang Cui, Krishnan V. Ramanujan, Tatsuaki Takeda, Yoshihisa Kitamura, Toshiaki Sendo, Ramachandran Murali. Constitutive NF-kB-activation loop enhances resistance to trastuzumab in HER2 positive luminal B breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 726. doi:10.1158/1538-7445.AM2015-726
Abstract 730: The role of ZEB1/ZEB2 and βIII-tubulin in mediating docetaxel-resistant prostate cancer
Authors: Hanrahan Karen , Prencipe Maria , Bugler Jane , Murphy Lisa , O'Neill Amanda , Watson R. William
Published: August 2015

Docetaxel therapy is the gold standard treatment for advanced castrate-resistant prostate cancer (CRPC). However, patients either do not respond or develop resistance over time. Transcriptomic and proteomic analysis of docetaxel-resistant prostate cancer sub-lines developed by our group revealed multiple mechanisms of resistance in line with advanced disease, including over-expression of anti-apoptotic proteins and alterations of NF-KB activation. The sub-lines also demonstrated a coordinated loss and gain of epithelial and mesenchymal markers respectively; a process characteristic of Epithelial-Mesenchymal Transition (EMT). Studies have highlighted a role of EMT in prostate cancer progression, metastasis and docetaxel resistance. However, the role of EMT drivers in mediating resistance is not defined. We hypothesise EMT to be a central mechanism of apoptotic resistance in advanced docetaxel-resistant prostate cancer, representing a target for therapeutic manipulation.

EMT was characterised in the PC-3 D12 and DU145 R docetaxel-resistant sub-lines through an increased invasive capacity, MMP-1 secretion and protein expression of E-cadherin transcriptional repressors ZEB1 and ZEB2, in comparison to parental cell lines. This was associated with an increased expression of βIII-tubulin; a tubulin isotype linked to taxane resistance and tumour aggressiveness. Upon treatment with the apoptotic trigger docetaxel (20nM), the PC-3 D12 and DU145 R sub-lines demonstrated significant resistance compared to parental controls. Simultaneous siRNA knockdown of ZEB1 and ZEB2 resulted in both a partial reversal in apoptotic resistance and a decrease in cell viability; this was also associated with a down-regulation of βIII-tubulin and a re-expression of E-cadherin.

Our results provide evidence of the process of EMT in in vitro models of docetaxel-resistant prostate cancer, which is associated with differential susceptibility to docetaxel and is partially reversed through siRNA knockdown of the EMT drivers, ZEB1 and ZEB2. In addition, we have identified a link between EMT and βIII-tubulin in our models of docetaxel resistance. Current experiments are investigating the tissue expression of ZEB1 and βIII-tubulin in prostate cancer metastases following docetaxel therapy, which will determine the clinical relevance of EMT and βIII-tubulin as mediators of docetaxel resistance in CRPC.

Citation Format: Karen Hanrahan, Maria Prencipe, Jane Bugler, Lisa Murphy, Amanda O'Neill, R. William Watson. The role of ZEB1/ZEB2 and βIII-tubulin in mediating docetaxel-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 730. doi:10.1158/1538-7445.AM2015-730
Abstract 733: A targetable GATA2-IGF2 axis confers aggressiveness in lethal prostate cancer
Authors: Vidal Samuel J. , Rodriguez-Bravo Veronica , Quinn Aidan , Williams Estrelania , de la Iglesia-Vicente Janis , Sun Xiaochen , Chen Xintong , Hoshida Yujin , Cordon-Cardo Carlos , Domingo-Domenech Josep
Published: August 2015

Castration resistant prostate cancer (CRPC) is a significant cause of mortality worldwide. In recent years, treatment modalities that improve survival have emerged including taxane chemotherapy and second generation androgen signaling inhibitors, among others. Indeed, today the first line chemotherapeutic docetaxel as well as the second line agent cabazitaxel are mainstays of treatment. However, CRPC inexorably progresses to a chemotherapy resistant state that ultimately precedes lethality. Elucidating the molecular determinants of aggressiveness in chemotherapy resistant CRPC may therefore stimulate new therapeutic strategies that improve clinical outcomes. We used laboratory models and clinical databases to identify GATA2 as a regulator of chemotherapy resistance and tumorigenicity in this context. Whole genome expression profiling, clinical validation and genetic screening approaches revealed that GATA2 regulates a consensus signature of cancer progression associated genes. Mechanistically, direct upregulation of the growth hormone IGF2 emerged as a significant mediator of the aggressive properties regulated by GATA2. IGF2 in turn activated IGF1R and INSR as well as a downstream polykinase program. The characterization of this axis prompted a novel combination strategy whereby dual IGF1R/INSR inhibition restored the efficacy of chemotherapy and improved survival in preclinical models. These studies reveal a GATA2-IGF2 aggressiveness axis in chemotherapy resistant prostate cancer and identify a therapeutic opportunity in this challenging disease.

Citation Format: Samuel J. Vidal, Veronica Rodriguez-Bravo, Aidan Quinn, Estrelania Williams, Janis de la Iglesia-Vicente, Xiaochen Sun, Xintong Chen, Yujin Hoshida, Carlos Cordon-Cardo, Josep Domingo-Domenech. A targetable GATA2-IGF2 axis confers aggressiveness in lethal prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 733. doi:10.1158/1538-7445.AM2015-733
Abstract 734: Synergistic antitumor effect of vemurafenib and metformin in thyroid cancer
Authors: Hanly Elyse K. , Tuli Neha Y. , Bednarczyk Robert B. , Moscatello Augustine L. , Darzynkiewicz Zbigniew , Geliebter Jan , Tiwari Raj K.
Published: August 2015

Advanced thyroid cancer patients such as those with radioiodine-refractory and metastatic differentiated thyroid cancer or anaplastic thyroid cancer lack effective treatment options. Targeted BRAF kinase inhibitors such as vemurafenib have shown promising results in clinical trials and off-label use for patients with BRAFV600E-positive tumors. While targeting BRAFV600E offers particular effectiveness and selectivity in inhibiting cancer cells in the short term, resistance does develop. Prior results in our laboratory confirmed that vemurafenib halts DNA synthesis and induces apoptosis in our mutated thyroid cancer cells. In addition, resistant BCPAP (papillary thyroid cancer) cells were developed in our laboratory by exposing cells to increasing concentrations of vemurafenib. Resistant cells were characterized by hyperactive mitogen-activated protein kinase (MAPK) signaling and resistance to mammalian target of rapamycin (mTOR) inhibition. These additional targets mediating resistance provide an opportunity to devise novel synergistic combination therapies. Since antitumor effects of the widely used antidiabetic agent metformin were observed in thyroid cancer cells and the drug is thought to suppress mTOR, we investigated metformin adjuvant therapy in combination with vemurafenib. Various cell lines including Nthy-ori 3-1 (normal immortalized thyroid cells), BCPAP (papillary thyroid cancer), vemurafenib-resistant BCPAP, and 8505c (anaplastic thyroid cancer) were analyzed in terms of cellular viability, apoptosis and expression of cell signaling molecules after exposure to both drugs. We found that the combination of vemurafenib and metformin caused significantly decreased viability in vemurafenib-resistant BCPAP cells. Furthermore, synergistic apoptosis was observed in BCPAP papillary thyroid cancer cells and 8505c anaplastic thyroid cancer cells in response to the combination treatment. These effects appeared to be mediated by inhibition of both mTOR and MAPK signaling. Results support further investigation into the use of metformin in combination with targeted kinase inhibitors to treat advanced thyroid cancer patients.

Citation Format: Elyse K. Hanly, Neha Y. Tuli, Robert B. Bednarczyk, Augustine L. Moscatello, Zbigniew Darzynkiewicz, Jan Geliebter, Raj K. Tiwari. Synergistic antitumor effect of vemurafenib and metformin in thyroid cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 734. doi:10.1158/1538-7445.AM2015-734
Abstract 738: Combined inhibition of non-canonical HER2 signaling as a strategy to overcome resistance to HER2-targeted therapies
Authors: Bernardi Ronald J. , Dominguez-Vidana Rocio , Bland Christopher S. , Scorsone Kathleen A. , Tyagi Siddhartha , Schmitt Earlene M. , Shea Martin J. , Mitchell Tamika , Rao Mitchell , Nanda Sarmistha , Hilsenbeck Susan G. , Shaw Chad , Osborne C. Kent , Schiff Rachel , Westbrook Thomas F.
Published: August 2015

Despite the availability of drugs directly targeting HER2, resistance to therapy remains a major clinical problem in HER2-positive breast cancer. Leveraging a series of RNAi genetic screens, we identify a novel signaling network governing response to HER2 inhibition in vitro and in vivo. We define WEE1 as a core component of this network. Genetic or pharmacologic inhibition of WEE1 or its upstream regulators kills HER2-positive breast cancer cells in combination with HER-family inhibitors. Mechanistically, we demonstrate a non-canonical role of HER2 in governing mitotic progression. Combined inhibition of WEE1 and HER2 abolishes this critical regulation, leading to mitotic catastrophe and apoptosis. Thus, we demonstrate an unexpected convergence of non-canonical HER2 signaling and WEE1 that can be exploited to overcome drug resistance. Our findings provide a strong mechanistic and pre-clinical rationale for combined HER2 and WEE1 inhibition as a treatment strategy for HER2-positive breast cancer.

Citation Format: Ronald J. Bernardi, Rocio Dominguez-Vidana, Christopher S. Bland, Kathleen A. Scorsone, Siddhartha Tyagi, Earlene M. Schmitt, Martin J. Shea, Tamika Mitchell, Mitchell Rao, Sarmistha Nanda, Susan G. Hilsenbeck, Chad Shaw, C. Kent Osborne, Rachel Schiff, Thomas F. Westbrook. Combined inhibition of non-canonical HER2 signaling as a strategy to overcome resistance to HER2-targeted therapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 738. doi:10.1158/1538-7445.AM2015-738
Abstract 747: Glutamine, glutaminase and γ-glutamyl-transferase activities are essential for lung tumorigenesis
Authors: Sappington Daniel R. , Siegel Eric R. , Penney Rosalind B. , Boysen Gunnar
Published: August 2015

Increased glutamine uptake has been found to drive cancer cell proliferation, making tumor cells glutamine-dependent or even addicted. Glutamine is believed to provide additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, a pathway known to be activated in tumor cells. Additionally, glutathione concentrations are known to be elevated in non-small cell lung cancer (NSCLC) and is believed to protect against oxidative stress. In contrast, glutathione's participation in the γ-glutamyl-cycle is less well studied. Here we investigated the hypothesis that glutamine drives glutathione synthesis and export, drives the γ-glutamyl-cycle, and thereby contributes to NSCLC tumor cell proliferation. Glutamine consumption and glutathione excretion were measured in H460, A549 and MRC-5 cell lines. Treatment with BPTES, a known GLS inhibitor, led to reduced cell proliferation. This was also demonstrated when γ-glutamyl-transferase (GGT) was inhibited with acivicin. GGT transfers glutamyl groups from glutathione to amino acids to facilitate the amino acid transport into the cell. These results suggest active utilization of the γ-glutamyl cycle in the tumor cells. The formation of metabolites downstream of GLS and GGT were essentially abolished at the lowest inhibitor concentrations studied. In contrast, biological endpoints, such as MTT assay response, required up to 30-fold higher inhibitor concentrations. Culturing in stable isotope-labeled glutamine demonstrated that >50% of excreted glutathione and >80% of γ-glutamyl amino acids were derived from glutamine. Together these results demonstrate that glutamine is actively utilized for synthesis and excretion of glutathione which, in turn, is utilized to promote amino acid uptake by the formation of γ-glutamyl amino acids. These findings add yet another important function to the glutamine dependence of tumor cells. In addition to providing TCA cycle intermediates, we show that glutamine promotes glutathione synthesis to increase cellular defense mechanisms, and drives the γ-glutamyl cycle to enhance amino acid uptake.

Citation Format: Daniel R. Sappington, Eric R. Siegel, Rosalind B. Penney, Gunnar Boysen. Glutamine, glutaminase and γ-glutamyl-transferase activities are essential for lung tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 747. doi:10.1158/1538-7445.AM2015-747
Abstract 76: Molecular profiling of AML patient derived xenograft models with deep sequencing using a 109 AML associated gene panel and a 409 gene comprehensive cancer panel
Authors: Huang Stephen , Lira Paul , Wang Kai , Zhang Cathy , Jackson-Fisher Amy , Ching Keith , Rejto Paul
Published: August 2015

We have developed and validated a 109 AML associated gene panel NGS assay based on sequence capture technologies. The overall assay sensitivity was 99.%, and overall assay specificity was 100% when sequenced with reference “gold” standard NA12878. Assay analytical accuracy was evaluated with diluted cell line samples harboring mutations in this gene panel, all diluted mutations with minor allele frequencies were detected, analytical limit of detection (LOD) is < 5%, and assay reproducibility is 96.8%. The assay also demonstrated robustness in sequencing 10 commercial AML patient samples (whole blood and bone marrow) and detecting mutations. Therefore, the AML gene panel deep sequencing assay is sensitive and accurate and is ready to be used for preclinical and clinical studies.

We subsequently sequenced 15 AML patient derived xenograft (PDX) models with this assay. These models were also deep sequenced with a 409 comprehensive cancer gene panel based on multiplex amplicon technologies. The mutation profiles of each model will be summarized in this presentation using two sequencing platforms. By analysis of the mutation profile of each model, sub-clones of mutations were revealed in the early passages of these models. Two of these models with FLT-3 mutations were later treated with Quizartinib and Crenolanib separately, the treated mouse models were profiled with these two targeted gene panels; clonal evolution of mutations in these models treated with FLT-3 inhibitors was documented.

Deep sequencing with target gene panel is a powerful tool to decipher the mutation sub-clones in the early passages of PDX models, and understand the acquired resistance of drug treatment.

Citation Format: Stephen Huang, Paul Lira, Kai Wang, Cathy Zhang, Amy Jackson-Fisher, Keith Ching, Paul Rejto. Molecular profiling of AML patient derived xenograft models with deep sequencing using a 109 AML associated gene panel and a 409 gene comprehensive cancer panel. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 76. doi:10.1158/1538-7445.AM2015-76
Abstract 760: On the evolution of erlotinib-resistant NSCLC subpopulations
Authors: Ramirez Michael E. , Steininger Robert J. , Wu Lani F. , Altschuler Steven J.
Published: August 2015

One of the foremost challenges to cancer treatment is the emergence of drug resistance. Adding complexity to this problem is the recent realization that drug resistance can stem from selection for both intrinsically resistant subpopulations as well as subpopulations that adapt to the drug challenge through epigenetic and/or signaling state changes. Because of this complexity, cells have many ways to survive in response to targeted inhibitors, resulting in a diversity of resistance states between and even within patient tumors. Ongoing strategies are aimed at finding the best second-line therapies for resistant tumors. However, the extent to which drug-resistant subpopulations exhibit similar or divergent sensitivities to second-line treatments is unknown. How different are drug-resistant subpopulations from one another? Do these differences affect the types of inhibitors that will be effective in killing these drug-resistant colonies, resulting in a variety of strategies for managing drug-resistant populations? What are the molecular changes that correspond to differences in secondary drug response? Here, we aim to determine the degree of heterogeneity between drug-resistant clonal populations by profiling their response to a panel of second-line therapies and examining mutational states to secondary drug response. This study will provide insight into whether or not drug-resistant subpopulations adopt similar or disparate drug-resistant states.

Citation Format: Michael E. Ramirez, Robert J. Steininger, Lani F. Wu, Steven J. Altschuler. On the evolution of erlotinib-resistant NSCLC subpopulations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 760. doi:10.1158/1538-7445.AM2015-760
Abstract 77: Genomic characterization of aggressive subtype of neuroblastoma without MYCN amplification
Authors: Ohira Miki , Tatsuno Kenji , Tsutsumi Shuichi , Yamamoto Shogo , Nakamura Yohko , Kamijo Takehiko , Aburatani Hiroyuki , Nakagawara Akira
Published: August 2015

Neuroblastoma is known to exhibit wide ranges of clinical behavior, from spontaneous regression to highly resistant to chemotherapy, and one of the challenges in the clinic is to develop adequate therapeutic strategies for the intermediate risk-type of patients (stage 3 or 4 without MYCN amplification) whose prognosis sometimes tends to be poor in long-time follow-up (10 years survival rate (SR) was 51% in our database, n = 161). To discover the key target genes or pathways for the unfavorable subset in this patient group, we have analyzed 44 primary tumors (dead:24, alive:20) without MYCN amplification nor ALK alteration by integrated genomic analyses such as array CGH, gene expression profiling and whole exome sequencing (SureSelect 50Mb, average read depth:157, x20 coverage:95%). We found 816 non-synonymous somatic single nucleotide variations (SNVs) in coding genes and alteration number in each tumor strongly associates with age at diagnosis (p<0.0001) and patient prognosis (p = 0.187). A novel ATRX mutation was identified in a tumor with 1p and 11q losses isolated from a male patient (age:93 months). Recurrent SNVs (n = 2 to n = 6) were found in 44 genes, 20 of those were involved only in the tumors from the patient with poor prognosis, in the 44 tumor set. Validation of these 20 SNVs is ongoing by using additional clinical samples. These features of genomic alterations could be useful for risk classification for each tumor and constructing new therapeutic strategies for aggressive neuroblastomas without MYCN amplification.

Citation Format: Miki Ohira, Kenji Tatsuno, Shuichi Tsutsumi, Shogo Yamamoto, Yohko Nakamura, Takehiko Kamijo, Hiroyuki Aburatani, Akira Nakagawara. Genomic characterization of aggressive subtype of neuroblastoma without MYCN amplification. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 77. doi:10.1158/1538-7445.AM2015-77
Abstract 771: Preclinical characterization of the selective FGFR inhibitor INCB054828
Authors: Liu Phillip CC , Wu Liangxing , Koblish Holly , Bowman Kevin , Zhang Yue , Klabe Ronald , Leffet Lynn , DiMatteo Darlise , Rupar Mark , Gallagher Karen , Hansbury Michael , Zhang Colin , He Chunhong , Collier Paul , Covington Maryanne , Wynn Richard , Yeleswaram Swamy , Vaddi Kris , Burn Timothy , Yao Wenqing , Huber Reid , Scherle Peggy , Hollis Gregory
Published: August 2015

Aberrant signaling through Fibroblast Growth Factor Receptors (FGFR) has been reported in multiple types of human cancers. Genomic analyses of squamous cell lung, gastric and urothelial tumors have revealed recurrent genetic alterations in FGFR1, FGFR2 and FGFR3 genes, respectively. FGFR proteins contribute to the development of malignancies by promoting tumor cell proliferation, survival, and migration and supporting angiogenesis. Therefore targeting FGFR kinases may provide therapeutic benefit to patients with cancers that have genetic alterations in genes encoding components of the FGF-FGFR axis. INCB054828 is a potent inhibitor of FGFR1, FGFR2, and FGFR3 that has selective pharmacological activity against cancer cells with FGFR alterations.

In vitro, INCB054828 potently inhibited the kinase activity of recombinant FGFR1, FGFR2 and FGFR3 enzymes and was highly selective against a panel of kinases including VEGFR2. In cellular assays, INCB054828 inhibited the autophosphorylation of FGFR proteins with low nanomolar IC50 values and blocked signal transduction by FGFR to downstream markers of pathway activation. Cancer cell lines that have genetic alterations in FGFR1, FGFR2 and FGFR3 were uniquely sensitive to growth inhibition by INCB054828, with IC50 values generally in the range of 3-50 nM, compared with cancer cell lines or normal cells without FGFR dependence (IC50 > 1500 nM). In vivo, once-daily oral administration of INCB054828 inhibited the growth of tumors that are dependent upon FGFR1, FGFR2 and FGFR3 activity at tolerated doses. Suppression of tumor growth was dose-dependent and correlated with pharmacodynamic inhibition of FGFR. Collectively, these preclinical studies demonstrate that INCB054828 potently and selectively inhibits models of FGFR-dependent cancers in vitro and in vivo, supporting the compound's clinical evaluation in patients harboring oncogenic FGFR activation.

Citation Format: Phillip CC Liu, Liangxing Wu, Holly Koblish, Kevin Bowman, Yue Zhang, Ronald Klabe, Lynn Leffet, Darlise DiMatteo, Mark Rupar, Karen Gallagher, Michael Hansbury, Colin Zhang, Chunhong He, Paul Collier, Maryanne Covington, Richard Wynn, Swamy Yeleswaram, Kris Vaddi, Timothy Burn, Wenqing Yao, Reid Huber, Peggy Scherle, Gregory Hollis. Preclinical characterization of the selective FGFR inhibitor INCB054828. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 771. doi:10.1158/1538-7445.AM2015-771
Abstract 773: Alectinib shows potent antitumor activity against both ALK- and RET-rearranged non-small cell lung cancers
Authors: Kodama Tatsushi , Tsukaguchi Toshiyuki , Satoh Yasuko , Yoshida Miyuki , Watanabe Yoshiaki , Kondoh Osamu , Sakamoto Hiroshi
Published: August 2015

We have shown that alectinib is a potent ALK inhibitor and is being evaluated in clinical trials for the treatment of ALK fusion-positive non-small cell lung cancer (NSCLC). Recently, RET and ROS1 fusion genes have been implicated as driver oncogenes in 1-2% of NSCLC and have been developed into promising molecular targets for antitumor agents. Here, we investigate the additional target indication of alectinib by testing its ability to inhibit the activity of RET and ROS1 kinases. In enzyme assay, alectinib showed kinase inhibitory activity against RET as wells as ALK but did not show against ROS1. Alectinib inhibited the growth of LC-2/ad cells harboring CCDC6-RET and Ba/F3 cells expressing KIF5B-RET by suppressing RET phosphorylation. Alectinib also showed antitumor activity in mouse models of RET fusion-positive tumors (LC-2/ad cells and Ba/F3 cells expressing KIF5B-RET) and of ALK fusion-positive tumors (NCI-H2228 cells harboring EML4-ALK). In addition, alectinib showed kinase inhibitory activity against RET gatekeeper mutations (RET V804L and V804M) and blocked KIF5B-RET gatekeeper mutation-driven cell growth. Our results suggest that alectinib is effective against RET fusion-positive tumors, as observed in ALK fusion-positive tumors. Thus, alectinib might be a therapeutic option for patients with RET fusion-positive NSCLC.

Citation Format: Tatsushi Kodama, Toshiyuki Tsukaguchi, Yasuko Satoh, Miyuki Yoshida, Yoshiaki Watanabe, Osamu Kondoh, Hiroshi Sakamoto. Alectinib shows potent antitumor activity against both ALK- and RET-rearranged non-small cell lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 773. doi:10.1158/1538-7445.AM2015-773
Abstract 79: An androgen response element-based reporter assay for the detection of androgen receptor activity in prostate cells
Authors: Azeem Waqas , Hellem Margrete R. , Olsen Jan R. , Hua Yaping , Marvyin Kristo , Li Lisha , Qu Yi , Lin Biaoyang , Ke Xisong , Oyan Anne M. , Kalland Karl-Henning
Published: August 2015

Background: The androgen receptor (AR) transcription factor plays pivotal roles in the development and progression of prostate cancer. AR is activated and maintained throughout prostate cancer progression even in castration-resistant prostate cancer (CRPC). AR and its classical target genes such as PSA (KLK3) are, however, efficiently shut off in basal epithelial prostate cells and possibly in prostate cancer stem cells (CSCs). There is limited information on the mechanisms that keep the AR OFF in these cells, and the conditions that turn the AR and target genes ON.

Results: Fluorescent and luminescent reporters were constructed to develop a functional assay for AR activity. These reporters contain the repeated AR binding promoter sequences and were designed in a way that the AR can activate these AR element (ARE) reporters. The 241B reporter was selected among the constructed ARE reporters on the basis of higher activity. The AR positive prostate cancer cell line, LNCaP, was transduced with the lentiviral 241B-mCherry fluorescence reporter. FACS analysis quantitated the ARE reporter activity when reporter cells were treated with androgen and with the AR antagonist, enzalutamide, or the anti-androgen, abiraterone. AR expression vectors were also constructed with the AR open reading frames cloned into lentiviral expression vectors. All constructs were sequenced to verify the correct AR DNA sequence and their activity were tested using ARE reporter system.

Conclusion: The developed ARE reporter system is useful to understand the mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells. It will help us to investigate the role of the AR in differentiation and proliferation of prostate cells and to study the AR activity in two and three dimensional cell cultures. This system can also be useful in screening for drugs with activity against the AR.

Citation Format: Waqas Azeem, Margrete R. Hellem, Jan R. Olsen, Yaping Hua, Kristo Marvyin, Lisha Li, Yi Qu, Biaoyang Lin, Xisong Ke, Anne M. Oyan, Karl-Henning Kalland. An androgen response element-based reporter assay for the detection of androgen receptor activity in prostate cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 79. doi:10.1158/1538-7445.AM2015-79
Abstract 795: Dovitinib exerts potent antitumor effects in gastrointestinal stromal tumors
Authors: Hung Huynh T. , Ong Richard , Chow Pierce
Published: August 2015

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumours of the gastrointestinal (GI) tract. Although Imatinib is the current first-line therapy for patients not suitable for surgical resection, 10 - 20% of GIST patients taking Imatinib do not respond to this therapy and 40-50% of the patients develop acquired resistance to Imatinib following partial response or stable disease. Thus, novel treatments are needed for GIST. In the present study, four different patient-derived xenograft (PDX) GIST models were used to demonstrate that Imatinib effectively caused tumor regression in GIST26-0208A and GIST09-0210 models with the common exon 11 KIT W557-K558 mutation (Imatinib-sensitive models), inhibited tumor growth of GIST27-0510 model with Y568-I571 and P573-L576 deletion and delayed tumor growth in GIST27-0612 with wild-type KIT. Unlike Imatinib, Dovitinib effectively caused tumor regression in all four PDX models tested regardless of their KIT mutational status. On GIST27-0510 model, tumor regression was observed as early as day 3 of Dovitinib treatment and continued to approximately 22% of the initial tumor volume. Dovitinib provided longer progression-free period than Imatinib after a 21-day treatment cycle. In the GIST26-0208A model, signs of tumor regrowth were observed on day 2 for Imatinib and day 59 for Dovitinib following a 21-day treatment. Dovitinib treatment took approximately 3.7 - 5 times longer to reach initial tumor volume than Imatinib following a 21-day treatment. Utilizing micro-Positron Emission Tomography (mPET), both Imatinib and Dovitinib treatment showed decreased SUV and TLG readings after 8-day treatment in GIST26-0208A. A trend of increasing activity was observed in Imatinib but not in Dovitinib-treated tumors at day 11 and day 21 following an 8-day treatment, suggesting mPET could potentially be used to access tumor response to Dovitinib. In Imatinib/Dovitinib-sensitive models, Imatinib and Dovitinib induced significant reduction in expression of p-ERK1/2, Bim, p-AKT (Ser473) and p-Cdc2 at 72 hours post treatment. Although inhibition of p-c-Kit (Tyr568/570) was observed, higher levels of p-Histone H2A-X (Ser139) and Cleaved PARP expression were observed in Dovitinib than Imatinib treatment, suggesting that Dovitinib was more effective than Imatinib in inducing cell death. Our data showed that Dovitinib treatment is able to provide greater advantages over Imatinib in all four PDX models regardless of their KIT mutational status, suggesting that Dovitinib may offer an alternative treatment for GIST that are insensitive to Imatinib.

Citation Format: Huynh T. Hung, Richard Ong, Pierce Chow. Dovitinib exerts potent antitumor effects in gastrointestinal stromal tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 795. doi:10.1158/1538-7445.AM2015-795
Abstract 796: Alofanib, a novel allosteric FGFR2 inhibitor, shows potent antitumor activity in ovarian cancer with FGFR2 expression
Authors: Tjulandin Sergei , Byakhov Mikhail , Stepanova Evgenia , Khochenkov Dmitry , Harrison Daniel , Tsimafeyeu Ilya
Published: August 2015

Alofanib (formerly known as RPT835) is a novel allosteric FGFR2 inhibitor with activity in FGFR2-expressing triple-negative breast cancer [Tjulandin S, et al. San Antonio Breast Cancer Symposium 2014]. Early data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity [Cole C, et al. Cancer Biol Ther. 2010]. We investigated antitumor activity of alofanib in ovarian cancer in vitro and in vivo.

To assess the efficacy of alofanib on FGF-mediated cell proliferation, ovarian cancer (SKOV-3) FGFR2-expressing cells were incubated in a 96-well microculture plate and were treated with serially diluted RPT835. Basic FGF was added at a concentration of 25 ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Immunocompromised mice were used for xenotransplantation of FGFR2 high-expressing ovarian cancer cells (SCOV-3). Seventy animals with measurable tumors were selected on day 10 and randomized into control groups (no treatment or chemotherapy alone (paclitaxel + carboplatin) and treatment groups (alofanib orally or intravenously (different dose levels) in combination with chemotherapy). Measurements of tumor volume (mm3) were performed by digital calipers every 3 days during 30 days after tumor inoculation.

Basic FGF significantly increased proliferation of the ovarian cancer cells in untreated control group (P = 0.001). Alofanib treatment resulted in growth inhibition of SKOV-3 cell line in vitro. Treatment of alofanib in combination with paclitaxel/carboplatin demonstrated significant tumor growth delay phenotype in all treatment groups compared to control non-treatment groups. Alofanib exhibited a dose-dependent effect on tumor growth. Daily intravenous regimen of alofanib (total maximum dose per week was 350 mg/kg) demonstrated dramatic effect (inhibiting growth by 80% and by 53% in comparison with vehicle and chemotherapy group alone, respectively (P<0.001). Alofanib decreased number of vessels in tumor on 49% compared with chemotherapy alone (P<0.05). There were tumor necrosis and cell degeneration in alofanib group.

These results provide strong rationale for evaluation of alofanib in combination with paclitaxel and carboplatin in patients with ovarian cancer.

Citation Format: Sergei Tjulandin, Mikhail Byakhov, Evgenia Stepanova, Dmitry Khochenkov, Daniel Harrison, Ilya Tsimafeyeu. Alofanib, a novel allosteric FGFR2 inhibitor, shows potent antitumor activity in ovarian cancer with FGFR2 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 796. doi:10.1158/1538-7445.AM2015-796
Abstract 799: Protein kinase C ϵ cooperates with Pten deficiency to regulate NF-κB pathway in prostate cancer progression
Authors: Garg Rachana , Blando Jorge , Perez Carlos J. , Benavides Fernando J. , Kazanietz Marcelo G.
Published: August 2015

Prostate cancer is one of the most commonly diagnosed malignancies and the second leading cause of cancer-related deaths among men in the United States. Protein kinase C epsilon (PKCϵ), a member of the PKC family of phorbol ester/diacylglycerol receptors, has emerged as an oncogenic kinase and shown to be up-regulated in human prostate cancer specimens. We recently demonstrated that PKCϵ is an upstream regulator of NF-κB activation in prostate cancer (JBC, 287:37570-37582, 2012) and that transgenic overexpression of PKCϵ in mice prostate under the control of the probasin promoter (PB-PKCϵ) leads to hyperplasia and PIN lesions but was insufficient to drive neoplastic changes (Cell Cycle, 10:268-277, 2011). Notably, when we intercrossed PB-PKCϵ mice with mice haploinsufficient for Pten, another common genetic alteration in human prostate cancer, the resulting compound mutant mice (PB-PKCϵ;Pten+/- mice) developed fully invasive adenocarcinoma with elevated NF-κB levels. In the present study, we aim to delineate the mechanism underlying the observed cooperativity between PKCϵ overexpression and Pten deficiency and to explore the consequences of this cooperativity on the transcription factor NF-κB signaling, a pathway known to be highly dysregulated in prostate tumorigenesis. To this end, we stably overexpressed PKCϵ in mouse prostate epithelial lines that are either heterozygous (P8) or homozygous (CaP8) for Pten deletion. We observed a striking synergism between PKCϵϵ overexpression and Pten loss in conferring enhanced proliferative, migratory and invasive phenotype. Moreover, LPS or TNFα stimulation of these cells led to increased NF-κB activation as evident from the elevated IκBα phosphorylation, NF-κB nuclear translocation and transactivation of a NF-κB luciferase reporter. These effects were much more pronounced in CaP8 cells. Of note, PKCϵ overexpression and Pten loss also cooperates to augment levels of the NF-κB regulated gene, COX-2. Stable overexpression of PKCϵ and Pten depletion in “normal” immortalized RWPE1 cells also resulted in significant enhancements in TNFα-induced NF-κB activation and COX-2 induction. Furthermore, NF-κB inhibition by parthenolide significantly retarded the growth of CaP8-PKCϵ tumors in athymic nude mice. Overall, our results identify NF-κB as a mediator of PKCϵ oncogenesis in prostate cancer, particularly in the context of Pten loss.

Citation Format: Rachana Garg, Jorge Blando, Carlos J. Perez, Fernando J. Benavides, Marcelo G. Kazanietz. Protein kinase C ϵ cooperates with Pten deficiency to regulate NF-κB pathway in prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 799. doi:10.1158/1538-7445.AM2015-799
Abstract 800: Dysregulation of DLL4 gene expression in Li-Fraumeni syndrome and tumorigenesis
Authors: Yao Zhixing , Sherif Zaki A.
Published: August 2015

Li-Fraumeni Syndrome (LFS), a clinically and genetically rare heterogeneous cancer syndrome, is characterized primarily by a p53 (TP53) gene mutation. This study is an attempt to understand why 30-40% of LFS patients who do not harbor p53 mutation are nonetheless besieged by the same preponderance of primary cancers, die at an early age, and at the same rate as those with the p53 mutation. LFS provides powerful insights into our understanding of the somatic mutations present in sporadic cancers influenced by p53. The p53 protein is a transcription factor that guards against genomic instability by inducing target genes that mediate its functions. Utilizing cytogenetic techniques, we previously discovered a novel balanced reciprocal translocation t(11;15)(q23;q15) between chromosomes 11 and 15 in the normal skin fibroblasts of a patient with LFS who had bilateral breast cancer but was homozygous for wild-type p53. Analysis of the breakpoint regions of this translocation yielded a number of genes; of particular interest was DLL4 (Delta-like ligand 4, locus 15q15.1), which encodes a ligand that binds to Notch receptors. Notch signaling controls cell fate specification during embryonic and postnatal development. DLL4 is a putative regulator of T cell lineage and thymopoiesis. In this study, we analyzed DLL4 gene expression and regulation in tumorigenesis. We also analyzed the transcriptional regulation of the DLL4 promoter. Our data reveal that DLL4 expression is abrogated in the normal skin fibroblasts of all LFS patients under study and markedly down-regulated in breast cancer cells and neuroblastoma cells due to TP53 mutation, DNA methylation or gene disruption caused by a balanced reciprocal translocation between chromosomes 11q23 and 15q15. Immunohistochemistry (IHC) staining corroborated the down-regulation of DLL4 in cancer tissue samples from different organs. These findings taken together not only have possible implications for karyotype-phenotype correlation in carcinogenesis, specifically of breast cancer, but also shed light on the possible implication of DLL4 in LFS and breast cancer initiation and progression. The drastic reduction or absence in the expression of DLL4 in normal skin fibroblasts of LFS patients as well as breast and brain cancer cells is significant and supports the concept that this ligand may play a role in cancer immune-surveillance and its resuscitation in the tumor microenvironment may stimulate T-cell immunity and suppress tumor growth. This demonstrates that DLL4 may provide a strong platform as an immuno-therapeutic target in LFS and breast cancer patients.

Citation Format: Zhixing Yao, Zaki A. Sherif. Dysregulation of DLL4 gene expression in Li-Fraumeni syndrome and tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 800. doi:10.1158/1538-7445.AM2015-800