bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4931: A circulating cell free DNA workflow for targeted sequencing - from isolation through validation
Authors: Dhingra Dalia , Fang Xingwang , Carter Matt , Brinza Dumitru , Scafe Charles , Davies Christopher , Hyland Fiona
Published: August 2015

Circulating cell free DNA has been shown to have the potential as a non-invasive substrate for the detection of cancer and its progression as well as the determination of therapeutic resistance. As circulating tumor DNA is often present at low frequencies within circulating cell free DNA, targeted sequencing is an optimal tool for mutation detection. Here, we demonstrate a complete workflow from isolation through validation of circulating tumor DNA.

We have optimized a protocol using magnetic beads to isolate circulating cell free DNA. This protocol is easily automated to process up to 192 samples a day. It is also easily scalable for any input volume and can elute in volumes down to 15 μL resulting in no loss of low frequency alleles. We demonstrate comparable performance between this bead based isolation and column based isolation.

We then completed molecular characterization of the isolated circulating cell free DNA using the multiplexing capabilities of AmpliSeq™ and the Ion PGM™. With the Ion AmpliSeq™ Cancer Hotspot Panel v2, we performed targeted sequencing of 50 genes of interest, covering 2800 COSMIC mutations. We demonstrate good reproducibility of amplicon representation as well as allelic frequencies. Through saturation studies and subsampling, we have determined the limit of detection of hotspots circulating cell free DNA on the Ion PGM™ to be below 1%. We further demonstrate proof of principle of this workflow on circulating cell free DNA and matched FFPE samples complete with TaqMan® validation.

Our results validate the accuracy and ease of our workflow. This protocol, from isolation through targeted sequencing and validation, will not only result in a simple sample preparation for circulating cell free DNA but also facilitate rapid mutation detection to advance cancer research.

Citation Format: Dalia Dhingra, Xingwang Fang, Matt Carter, Dumitru Brinza, Charles Scafe, Christopher Davies, Fiona Hyland. A circulating cell free DNA workflow for targeted sequencing - from isolation through validation. [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 4931. doi:10.1158/1538-7445.AM2015-4931
Abstract 4938: APC in breast cancer: the ABCs of gene expression
Authors: Fernandez Soto Katia , VanKlompenberg Monica K. , Cole Jennifer , Prosperi Jenifer R.
Published: August 2015

Breast cancer is the most common malignancy and cancer-related cause of death in non-smoking women in the United States. Due to the heterogeneity of this disease, the oncogenic and signaling pathways contributing to these tumors are distinct. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in up to 70% of sporadic breast cancers; however, the mechanism by which APC mutation impacts tumorigenesis remains unexplored. Up-regulation of the zinc finger transcription factor EGR-1 has been associated with cell invasion and enhanced drug resistance, which is an important property of cancer stem cells (CSCs). ALDH1 and STAT3 have been useful in the identification of CSCs, which are associated with poor clinical outcomes including increased tumor survival and invasion. We utilized two human breast cancer cell lines, MDA-MB-157 and DU4475, and cells isolated from the Mouse Mammary Tumor Virus-Polyoma middle T (MMTV-PyMT);ApcMin/+ mouse model. APC was knocked down in the MDA-MB-157 cells through shRNA lentiviral transduction, or transfected into DU4475 cells. First we looked at the expression of the transcription factor EGR-1 through Real-Time PCR in the MDA-MB-157 and DU4475 cell lines. Next, ALDH1 and STAT3 were measured in the MMTV-PyMT;ApcMin/+ mouse model and in MDA-MB-157 cells through western blots. In addition, an ALDEFLUOR assay was used to measure ALDH1 activity in the mouse model. To translate our findings to an in vivo model, we injected MMTV-PyMT;ApcMin/+ cells into the mammary fat pads of FVB-N syngeneic mice. EGR-1 expression was increased in both MDA-MB-157 APC-knockdown and DU4475 APC-mutant cells compared to cells expressing APC. In addition, STAT3 expression was found to be increased in MDA-MB-157 APC-knockdown and MMTV-PyMT;ApcMin/+ cells compared to MDA-MB-157 APC-wildtype and MMTV-PyMT;Apc+/+ cells. ALDH1 activity and tumor development were enhanced in ApcMin/+ cells compared to control cells. Combined, these data suggest that APC knockdown increases tumorigenic properties of cancer cells through EGR-1 overexpression and modulation of known cancer stem cell markers, ALDH1 and STAT3. Future studies will include measuring the expression of EGR-1 in the MMTV-PyMT;ApcMin/+ cells as well as ALDH1 in the human cell lines. Furthermore, because of the overexpression of these genes and markers, strategies designed to target positive populations might lead to more effective therapies of APC-mutant breast cancers.

Citation Format: Katia Fernandez Soto, Monica K. VanKlompenberg, Jennifer Cole, Jenifer R. Prosperi. APC in breast cancer: the ABCs of gene 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 4938. doi:10.1158/1538-7445.AM2015-4938
Abstract 4940: Role of Smad3 linker phosphorylation in breast cancer progression
Authors: Liu Fang , Xie Wen , Liu Zhengxue , Roy Tanima , Bae Eunjin , Wakefield Lalage M. , Kim Seong-Jin , Ooshima Akira , Reiss Michael , Matsuura Isao
Published: August 2015

Smad3 plays an important role in inhibiting cell proliferation and promoting apoptosis in multiple tissues, including the breast tissue. At late stages, Smad3 promotes breast cancer progression and metastasis. We previously showed that the proline-rich linker region of Smad3 is phosphorylated by several kinases, such as by CDKs and MAP kinases. We have mapped these phosphorylation sites. We show that mutation of the phosphorylation sites in the Smad3 linker region increases the ability of Smad3 to inhibit cell proliferation and promote apoptosis. Interestingly, mutation of the Smad3 linker phosphorylation sites also increases the ability of Smad3 to activate genes that promote metastasis. Accordingly, mutation of the Smad3 linker phosphorylation sites markedly inhibits tumorigenicity but promotes metastasis of breast cancer cell lines. Using human breast cancer tissue microarrays, we further show that Smad3 linker phosphorylation is progressively reduced along breast cancer progression. Taken together, our findings strongly suggest that Smad3 linker phosphorylation promotes tumorigenesis but inhibits metastasis. Our findings have important implications for cancer therapy.

Citation Format: Fang Liu, Wen Xie, Zhengxue Liu, Tanima Roy, Eunjin Bae, Lalage M. Wakefield, Seong-Jin Kim, Akira Ooshima, Michael Reiss, Isao Matsuura. Role of Smad3 linker phosphorylation in breast 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 4940. doi:10.1158/1538-7445.AM2015-4940
Abstract 4951: Expression analysis of the LOT1 gene in head and neck cancer
Authors: EKIZOGLU ERATAK SEDA , Karaman Emin , BUYRU NUR A.
Published: August 2015

Head and neck cancer (HNC) which is the sixth most common type of cancer worlwide, comprise a diverse group of tumors including neoplasias of the paranasal sinuses, the oral cavity, the trachea, the pharynx and the larynx. The most well known environmental risk factors for developing HNC are tobacca, alcohol and betel nut use and sexually transmitted viral pathogens such as human papilloma viruses (HPV). In addition to environmental factors, genetic factors may play a key role in the development of HNC. Therofore, genetic abnormalities have been studied extensively.

The LOT1 (Lost-on-transformation 1) gene is localized on human chromosome 6q24-25, a chromosomal region that is frequently deleted in many types of human cancer including squamous cell carcinomas of the head and neck. The product of the LOT1 gene is a 463 amino acid protein which contains 7 C2H2-type zinc-finger motifs at the amino terminal, as well as proline and glutamine rich areas at the carboxyl terminal. Functional analysis of LOT1 demonstrated that it may play a significant role as a transcription factor modulating growth suppression through mitogenic signaling pathways.

To elucidate the role of the LOT1 gene in tumorigenesis, many studies analyzing alterations of the gene expression were performed. As a result of these studies, the absence or reduced expression of LOT1 gene was reported in many types of tumors including breast, ovarian, pituitary and lung tumors.

In this study, we analyzed the role of the LOT1 gene in head and neck carcinogenesis. For this purpose, we investigated the expression level of LOT1 gene in tumor tissue and adjacent non-cancerous tissue samples from 81 patients with HNC by quantitative RT-PCR.

We found that LOT1 gene expression was decreased in 64 of 81 tumor tissues (79%) when compared to normal tissue. Statistically, downregulation of the LOT1 gene mRNA expression was significant (p<0.001). No significant correlation was found between LOT1 gene expression and clinicopathological parameters including age, sex, stage, histological grade and tumor location (p>0.05).

Our data indicate that loss of the LOT1 gene function may play a role in the etiology of head and neck carcinogenesis.

Citation Format: SEDA EKIZOGLU ERATAK, Emin Karaman, NUR A. BUYRU. Expression analysis of the LOT1 gene in head and neck 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 4951. doi:10.1158/1538-7445.AM2015-4951
Abstract 4957: Next generation sequencing demonstrates association between tumor suppressor gene aberrations and poor outcome in patients with cancer
Authors: Schwaederle Maria , Daniels Gregory A. , Piccioni David E. , Fanta Paul T. , Schwab Richard B. , Shimabukuro Kelly A. , Parker Barbara A. , Kurzrock Razelle
Published: August 2015

Next generation sequencing is advancing at a breathtaking pace, and is transforming patient care by allowing physicians to customize and match treatment to their patients’ tumor alterations. Our goal was to study the association between key molecular alterations and outcome parameters. We retrospectively reviewed the clinicopathologic and clinical outcomes of 392 patients for whom molecular testing had been performed. Potential associations between clinical characteristics and outcome parameters (overall survival (OS), time to metastasis/recurrence, and best progression-free survival (PFS)) were examined. The Kaplan-Meier method and Cox regression models were used for our analysis, and results were subjected to internal validation using a resampling method (bootstrap analysis). In a multivariable analysis (Cox regression model), the parameters that were statistically associated with a poorer overall survival were the presence of metastases at diagnosis (P = 0.014), gastrointestinal histology (P<0.0001), PTEN (P<0.0001), and CDKN2A alterations (P = 0.0001). The variables associated with a shorter time to metastases/recurrence were gastrointestinal histology (P = 0.004), APC (P = 0.008), PTEN (P = 0.026) and TP53 (P = 0.044) alterations. TP53 (P = 0.003) and PTEN (P = 0.034) alterations were independent predictors of a shorter best PFS. A personalized treatment approach (matching the molecular aberration with a cognate targeted drug) also correlated with a longer best PFS (P = 0.046). Our study demonstrated that, across diverse cancers, anomalies in specific tumor suppressor genes (PTEN, CDKN2A, APC, and/or TP53) were independently associated with a worse outcome, as reflected by time to metastases/recurrence, best PFS on treatment, and/or overall survival. These observations suggest that molecular diagnostic tests may provide important prognostic information in patients with cancer.

Citation Format: Maria Schwaederle, Gregory A. Daniels, David E. Piccioni, Paul T. Fanta, Richard B. Schwab, Kelly A. Shimabukuro, Barbara A. Parker, Razelle Kurzrock. Next generation sequencing demonstrates association between tumor suppressor gene aberrations and poor outcome in patients with 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 4957. doi:10.1158/1538-7445.AM2015-4957
Abstract 496: Checkpoint kinase 1 inhibition suppresses cell growth and enhances cisplatin sensitivity in medulloblastoma cells
Authors: Shah Monil , Prince Eric , Venkataraman Sujatha , Balakrishnan Ilango , Alimova Irina , Harris Peter , Remeke Marc , Taylor Michael D. , Handler Michael H. , Foreman Nicholas K. , Vibhakar Rajeev
Published: August 2015

Background: Medulloblastoma is the most common malignant brain tumor in children and remains a therapeutic challenge due to its significant therapy-related morbidity. Checkpoint kinase 1 (CHK1) is highly expressed in many cancers and regulates critical steps in mitotic progression and DNA-damage response. Activation of CHK1 pathway promotes treatment resistance in tumor cells. Recent studies suggest that targeting CHK1 with a small molecule inhibitor, to sensitize tumors to a variety of DNA-damaging agents, is a promising approach to tumor therapy.

Methods: The expression of CHK1 mRNA in medulloblastoma patient samples and cell lines were examined using microarray analysis and qRT-PCR respectively. Western blot analysis was conducted on medulloblastoma cell lines to analyze expression level of CHK1 protein. The effect of AZD7762, a small molecule inhibitor of CHK1, on colony forming ability of medulloblastoma cell lines in combination with cisplatin was examined using colony formation assay and methylcellulose assay. The impact of AZD7762 in combination with cisplatin on tumor cell proliferation was further evaluated in real-time using xCELLigence. The effect of AZD7762 on cell viability was assessed using Guava ViaCount assay. Furthermore, the effects on key signaling pathways were analyzed using immunofluorescence, senescence assay, and apoptosis assay.

Results: Analysis of gene expression and western blot experiments revealed that CHK1 mRNA and protein levels are over expressed in all medulloblastoma patient samples and in cell lines when compared to normal pediatric cerebellum. High CHK1 expression correlates with adverse outcomes in a large cohort of medulloblastoma patients. Inhibition of CHK1 by a low nanomolar concentration of AZD7762 potently inhibited cell growth, suppressed the colony-forming ability, and increased cellular apoptosis of medulloblastoma cells. Furthermore, AZD7762 pretreatment sensitized medulloblastoma cells to cisplatin synergistically.

Conclusions: CHK1 expression is a prognostic marker in medulloblastoma and targeting CHK1 with a small molecule inhibitor, in combination with cisplatin, is an attractive strategy in treatment of medulloblastoma that warrants further investigation.

Citation Format: Monil Shah, Eric Prince, Sujatha Venkataraman, Ilango Balakrishnan, Irina Alimova, Peter Harris, Marc Remeke, Michael D. Taylor, Michael H. Handler, Nicholas K. Foreman, Rajeev Vibhakar. Checkpoint kinase 1 inhibition suppresses cell growth and enhances cisplatin sensitivity in medulloblastoma 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 496. doi:10.1158/1538-7445.AM2015-496
Abstract 4979: SIRT1 regulates the function of the KRAB-associated protein 1 (KAP1) in DNA damage response signaling
Authors: Lin Yi-Hui , Lou Zhenkun
Published: August 2015

KRAB-Associated Protein 1 (KAP1) is a master regulator for genome stability, heterochromatin formation, and target gene silencing. Many studies have suggested that post-translational modification is required for KAP1 to execute pleiotropic effects. Here, we have identified the physical interaction between KAP1 and SIRT1, which is a widely recognized NAD+-dependent deacetylase in DNA damage response. Therefore, the objective of this study was to characterize the functional significance of KAP1 deacetylation in DNA damage repair signaling.

In this study, SIRT1 was identified as a major interacting partner of KAP1 via biochemical approaches. Inhibition of SIRT1 activity by shRNA or selective inhibitor was able to upregulate the total acetylation level on KAP1, suggesting that KAP1 is a novel target of SIRT1. The major SIRT1-regulated KAP1 deacetylation sites (Lys266, Lys377, Lys469, and Lys770) were detected by quantitative mass spectrometric analysis. Next, to explore the functional significance of KAP1 deacetylation in NHEJ-mediated repair, KAP1 depleted cells reconstituted with wild-type KAP1 or 4KR acetylation mutant were used in the in vivo end-joining reporter system. Compare to wild-type KAP1, the 4KR mutant presented higher activity in promoting NHEJ efficiency. Furthermore, 4KR mutant showed stronger binding affinity to 53BP1, a crucial NHEJ promoting factor, and stabilized the 53BP1 foci formation in response to DNA damage.

In summary, this study unambiguously established KAP1 as a target of SIRT1 and that the deacetylation of KAP1 on Lys266, Lys377, Lys469, and Lys770 stabilizes the formation of 53BP1 foci on sites of double-strand breaks, thereby promotes NHEJ-mediated repair pathway.

Citation Format: Yi-Hui Lin, Zhenkun Lou. SIRT1 regulates the function of the KRAB-associated protein 1 (KAP1) in DNA damage response signaling. [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 4979. doi:10.1158/1538-7445.AM2015-4979
Abstract 4980: Rational design of combination therapies and block of acquired targeted drug resistance
Authors: Reuveni Hadas , Kuperschmidt Lana , Carmi Shani , Moskovitz Neta , Stemmer Salomon , Haviv Izhak
Published: August 2015

Recent successes of targeted drugs on end stage cancer patients highlight the value of mutation-based biomarkers with high positive predictive value for drug response predictions. However, the impact of these drugs is often temporary, and the patients progress to acquired resistance. A common mechanism of drug evasion involves feedback mechanisms that increase expression or phosphorylation-driven activation of an alternative oncogenic pathway. The objective of this work was to set up a streamlined methodology for pre-clinical assessment of the novel drug for refractory cancer targeting, with specific focus of personalized medicine, mediated by sequencing, and seek combination therapies that would block the spontaneous drug evasion. We explored the efficacy of the combination approach on a panel of cancer patient derived xenografts in mice, using tumor size, or metabolic imaging as an end point. Each cancer case was subject to target somatic mutation screening, which resulted in a targeted drug recommendation, and then, mouse groups were treated either with sequeincing-based therapy, or with combination of these therapies with blockers of the suspected evasion mechanisms. As a blocker of the evasion mechanism and epithelial to mesenchymal transition, we characterized the utility of a molecule that leads to the destruction of IRS1/2 of the IGF1R pathway. IRS1/2 are altered during EMT and drug resistance, thus blocking them could block drug resistance, and found multiple scenarios, where the combination of this blockage with currently used targeted drugs, such as Erlotinib, Zelboraf, and Gleevec, all increase the attenuation of the cancer growth and progression. Addition of the IRS1/2 destruction to Erlotinib and Zelboraf resistant H&N and melanoma cancers, respectively, lead to regression of the recurrent tumor mass. This combination therapy was further characterized by shRNA-negative screens, and kinome activity profiling, to assess further drug treatments that could be added, and in search for biomarkers that would identify patients likely to respond to the therapeutic combinations, We believe blocking the IGF1R in combination with drugs that inhibit the MAPK and tyrosine kinase receptor pathways may increase the progression free survival and expand the responding population within an array of cancer types, many of which are currently refractory to chemotherapy.

Citation Format: Hadas Reuveni, Lana Kuperschmidt, Shani Carmi, Neta Moskovitz, Salomon Stemmer, Izhak Haviv. Rational design of combination therapies and block of acquired targeted drug resistance. [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 4980. doi:10.1158/1538-7445.AM2015-4980
Abstract 4993: β-arrestin-2 regulates CXCR7-mediated EGFR transactivation and tumor cell proliferation in prostate cancer cells
Authors: Kallifatidis Georgios , Munoz Daniel , Singh Rajendra K. , Lokeshwar Bal L.
Published: August 2015

We have previously shown that the chemokine receptor CXCR7 trans-activates the epidermal growth factor receptor (EGFR) and is implicated in prostate tumor growth. However, the molecular mechanisms of CXCR7 mediated EGFR-trans-activation are not known. In the present work we show that CXCR7 induced EGFR transactivation is independent of EGF-like ligands such as amphiregulin and heparin-bound EGF. Importantly, treatment with pertussis toxin, a known G-protein inhibitor, had no effect on EGFR phosphorylation, indicating that G-protein signaling was not involved in CXCR7 mediated EGFR transactivation. Since CXCR7 is known to signal through β-arrestin-2 in its ligand dependent manner, we evaluated the role of β-arrestin-2 in CXCR7 induced EGFR trans-activation in prostate cancer cells. We used shRNA to stably down regulate β-arrestin-2 in LNCaP cells to evaluate the role of β-arrestin-2 in CXCR7 mediated induction of EGFR and downstream mitogenic signaling such as ERK1/2 and the phosphoinositide 3-kinase/Akt pathway. Interestingly, down regulation of β-arrestin-2 significantly induced phosphorylation of EGFR at Y1110 and Y1068 along with the phosphorylation/activation of ERK and AKT and an induction of proliferation/colony formation in prostate tumor cells. Moreover, shRNA mediated down regulation of β-arrestin-2 resulted in a decrease of p21 which is in accordance with the observed increased proliferation rate. The results of our study suggest that β-arrestin-2 is crucial negative regulator of CXCR7/EGFR signaling and underscore the importance of β-arrestin-2 in regulation of CXCR7/EGFR mediated tumor cell proliferation.

Citation Format: Georgios Kallifatidis, Daniel Munoz, Rajendra K. Singh, Bal L. Lokeshwar. β-arrestin-2 regulates CXCR7-mediated EGFR transactivation and tumor cell proliferation in prostate cancer 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 4993. doi:10.1158/1538-7445.AM2015-4993
Abstract 4994: RET isoforms differentially contribute to focal adhesion formation
Authors: Yoganathan Piriya , Lian Eric , Mulligan Lois M.
Published: August 2015

RET is a receptor tyrosine kinase crucial for the development of the kidney, and some neuroendocrine tissues. RET activation by its ligand, glial cell-line derived neurotrophic factor (GDNF), results in autophosphorylation of several key tyrosine kinase residues in the intracellular region, which allow binding of adaptor and signalling proteins, and consequently activation of various cellular processes, including survival, proliferation and migration. RET has two major protein isoforms, called RET9 and RET51 after the number of unique amino acids at the C-terminus, generated by alternative splicing. Although the RET isoforms are highly conserved across species, and both isoforms are normally co-expressed, they have major molecular and functional differences, including different transforming abilities, and trafficking properties. RET is implicated in several human diseases. Gain-of-function RET mutations result in the cancer syndrome multiple endocrine neoplasia type 2, that affects neuroendocrine tissues, somatic RET rearrangements are found in papillary thyroid carcinoma, and lung adenocarcinoma, and aberrant wild-type RET activation occurs in many cancers, including breast and pancreatic cancer. Focal adhesion protein complexes are involved in interactions of cells with the extracellular matrix, and are crucial for controlling cancer-related processes, including cell migration and invasion. The contributions of RET isoforms to the processes of focal adhesion formation and dynamics have not yet been investigated. To explore the roles of RET isoforms in focal adhesion formation, we used total internal resonance fluorescence (TIRF) microscopy to assess localization of various focal adhesion proteins, including paxillin, vinculin and zyxin in response to GDNF in a cell-based model system. Our results suggest that RET51 promotes more early (ie. paxillin) and late (ie. zyxin) stage focal adhesion formation than does RET9. Additionally, we demonstrate that the kinase dead form of RET51 results in a decrease of the number of focal adhesions compared to RET51 wild-type, suggesting that the increase in focal adhesion formation in the presence of RET51 is phosphorylation-dependent. Inhibition of SRC and FAK led to a reduction in focal adhesion formation, indicating that these pathways play roles in this process. Ongoing studies are investigating the focal adhesion dynamics of the RET isoforms and further characterizing the molecular differences underlying the ability of RET51 to induce greater focal adhesion formation than RET9.

Citation Format: Piriya Yoganathan, Eric Lian, Lois M. Mulligan. RET isoforms differentially contribute to focal adhesion formation. [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 4994. doi:10.1158/1538-7445.AM2015-4994
Abstract 4997: Phosphorylation of SLAP by the KIT-D816V oncogenic mutant suppresses SLAP-mediated negative regulation of KIT signaling
Authors: Kazi Julhash U. , Sun Jianmin , Rönnstrand Lars
Published: August 2015

The SRC-like-adaptor protein (SLAP) is a negative regulator of mitogenic signaling. SLAP shares considerable structural homology with SRC, apart from lacking a kinase domain. SLAP is known to be expressed in a variety of cells and regulates cell surface receptor signaling by direct association. Here we show that SLAP interacts with the type III receptor tyrosine kinase KIT and also with its oncogenic mutant KIT-D816V. This mutant has been described as a driving mutant in several cancers including mastocytosis and certain types of acute myeloid leukemia. The association is mediated through phosphotyrosine residues in KIT and the SLAP-SH2 domain. Despite high structural similarity between SLAP and SRC, these tyrosine residues are different from the ones mediating SRC binding to KIT. This association results in increased ubiquitination and degradation of KIT. siRNA-mediated depletion of SLAP selectively potentiates wild-type KIT downstream signaling but not KIT-D816V signaling. Although SLAP displays similar localization pattern with both ligand-stimulated KIT and KIT-D816V, only KIT-D816V can phosphorylate SLAP on three tyrosine residues (Y120, Y258 and Y273). This phosphorylation takes place when SLAP interacts with KIT-D816V. Mutations on these tyrosine residues in SLAP restores normal SLAP regulation on oncogenic KIT-D816V. Taken together, our data suggest that the oncogenic mutant of KIT can bypass normal cellular regulation of signaling by phosphorylating regulatory proteins such as SLAP.

Citation Format: Julhash U. Kazi, Jianmin Sun, Lars Rönnstrand. Phosphorylation of SLAP by the KIT-D816V oncogenic mutant suppresses SLAP-mediated negative regulation of KIT signaling. [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 4997. doi:10.1158/1538-7445.AM2015-4997
Abstract 5016: Short-term blockade of EGF signaling improves immune-mediated cytotoxicity of NSCLC cells
Authors: Dominguez Charli
Published: August 2015

One of the manifestations of tumor plasticity is the acquisition of mesenchymal-like features by epithelial tumor cells via the phenomenon of epithelial-mesenchymal transition (EMT). This phenotypic conversion of tumor cells is known to be associated with tumor resistance to multiple cancer therapies, including chemotherapy, radiation and some small molecule-targeted therapies. Recent work from our group demonstrated that tumor cells undergoing the EMT may also become resistant to immune-mediated cytotoxicity involving antigen-specific and innate effector cells. As signaling through the epidermal growth factor receptor (EGFR) is a major inducer of EMT in epithelial cells, in the present study we sought to investigate the effect of short-term EGFR inhibition with erlotinib on tumor phenotype, and provide rationale for combination with immune-mediated therapies. Our data shows that pretreatment with the EGFR TKI erlotinib for less than 72 hours modulates tumor plasticity and immune-mediated cytotoxicity in NSCLCs harboring a sensitizing EGFR mutation. A significant modulation in expression of multiple EMT markers can be observed, including significant changes in the levels of expression of the transcription factor brachyury, a driver of human tumor EMT and a target for immunotherapeutic interventions. In addition, significant modulation of immune-relevant molecules on the surface of tumor cells can be observed, including changes in the levels of TRAIL-receptors, co-stimulatory and checkpoint molecules. The observed phenotypic changes in response to short-term erlotinib treatment correlate with an enhancement of immune-mediated tumor cell lysis, without any observed adverse affect on effector cell populations. Taking into account that brachyury-based cancer vaccines are currently being evaluated in clinical trials, modulation of brachyury levels and the observed enhancement of immune-mediated lysis in erlotinib-treated tumors may provide the rationale for future combinations of erlotinib and brachyury-based vaccines for the treatment of lung carcinomas.

Citation Format: Charli Dominguez. Short-term blockade of EGF signaling improves immune-mediated cytotoxicity of NSCLC 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 5016. doi:10.1158/1538-7445.AM2015-5016
Abstract 5031: A humanized mouse model for translational assessment of targeted immune checkpoint blockade
Authors: Baia Gilson , Vasquez-Dunddel David , Ciznadija Daniel , Sidransky David , Katz Amanda , Paz Keren
Published: August 2015

The blockade of immune checkpoints is a promising therapeutic avenue for cancer therapy, with durable objective responses observed in patients with a variety of solid tumors. Despite these successes, current animal models do not reliably identify targets with the greatest clinical potential, due in part to differences between human and murine immune systems. Hence, robust preclinical tools to test these drugs directly against human cancers in the context of a human immune system are required. Champions Oncology has now developed the ImmunoGraft, whereby two innovative technologies, the patient-derived xenograft and humanized mice (immunodeficient mice reconstituted with a human immune system), are combined in a single platform.

Immune-compromised NOG (PrkdcscidIl2rgtm1Sug) mice were reconstituted (humanized) with human CD34+ cells and twelve weeks after humanization, mature human CD45+ cells comprised close to 50% of the leukocytes detected in the circulation and secondary lymphoid tissues of the now humanized animals. Growth of NSCLC tumors implanted into humanized NOG animals was comparable to those in non-humanized counterparts, and when humanized animals harboring NSCLC tumor implants were treated with ipilimumab, tumor growth was slowed, exhibiting marked regression. Systemic immune activation was observed in humanized mice following treatment with ipilimumab, characterized by robust proliferation of huCD3+ T cells and activated huCD4+ Th1 cells, as well as an increase in tumor-infiltrating huCD8+ cytotoxic T lymphocytes (all relative to vehicle controls).

We have developed an innovative new preclinical cancer model, the ImmunoGraft, which will enable novel immunotherapeutic agents to be accurately evaluated for efficacy against virtually any cancer indication that can be engrafted. As a proof-of-concept of the utility of the ImmunoGraft, we demonstrated how effectively ipilimumab restricted the growth of NSCLC implants engrafted in humanized animals. This translational platform is, therefore, more reflective of the human tumor microenvironment (both immune and non-immune cell-based) and is the most clinically-relevant model for screening immune system-targeting therapies, as single agents or in combination with standard-of-care drugs and other immuno-modulators. The ImmunoGraft has strong potential to revolutionize the application of immunotherapy to both personalized oncology and translational drug discovery.

Citation Format: Gilson Baia, David Vasquez-Dunddel, Daniel Ciznadija, David Sidransky, Amanda Katz, Keren Paz. A humanized mouse model for translational assessment of targeted immune checkpoint blockade. [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 5031. doi:10.1158/1538-7445.AM2015-5031
Abstract 5036: Tumor-induced activation of the Wnt/β-catenin/TCF4 pathway in dendritic cells promotes immune tolerance
Authors: Hong Yuan , Manoharan Indumathi , Suryawanshi Amol , Manicassamy Santhakumar
Published: August 2015

Tumors actively suppress host antitumor immune response and this represents a fundamental barrier to successful cancer immunotherapy. However, the molecular mechanisms underlying tumor-induced immune tolerance are largely unknown. In the present study, we show that Wnt ligands in the tumor microenvironment activate the β-catenin/TCF4 pathway within dendritic cells (DCs) rendering them tolerogenic and able to suppress host antitumor immunity against melanoma. This was because Wnt/β-catenin/TCF4 signaling in DCs induced retinoic acid synthesizing enzymes, which induced T regulatory responses and suppressed effector T cells. Consistent with this observation, DC-specific deletion of key downstream mediators of canonical Wnt signaling (Wnt co-receptors low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6), β-catenin and transcription factor 4 (TCF4)) in mice markedly delayed melanoma growth and enhanced host anti-tumor immunity. Likewise, pharmacological inhibition of the Wnt/β-catenin/TCF4 pathway in vivo had similar effects on tumor growth and effector T cell responses. Hence, Wnt/β-catenin/TCF4 pathway links together DCs, Tregs and the retionoic acid pathway, and is an important target for anticancer immunotherapy.

Note: This abstract was not presented at the meeting.

Citation Format: Yuan Hong, Indumathi Manoharan, Amol Suryawanshi, Santhakumar Manicassamy. Tumor-induced activation of the Wnt/β-catenin/TCF4 pathway in dendritic cells promotes immune tolerance. [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 5036. doi:10.1158/1538-7445.AM2015-5036
Abstract 5040: Calcium channels: Potential new therapeutic targets for hormone independent and resistant breast cancers
Authors: Cyrus Kedra , Kaushal Mudit , Sharawi Zeina , Noguchi Glyn , Chang Tiffany , Rydzewski William , Gibrel Fatima , Yeguech William , Haddad Bassam , Martin Mary Beth
Published: August 2015

Estrogen receptor alpha (ERα) is central to the growth and progression of a subset of breast cancers. As such, therapies that target ERα are a standard of care in the treatment of ERα -positive disease. Unfortunately, one-third of patients receiving hormonal therapies develop resistance. Several factors are believed to contribute to resistance including increased hormone independent activation of ERα by activated signal transduction pathways such as the epidermal growth factor (EGF) signaling pathway. We have shown that calcium mediates the cross talk between the EGF signaling pathway and ERα by directly binding to and activating the ligand binding domain of ERα suggesting a role for calcium in the development and progression of hormone independent and resistant breast cancer. Preliminary results from this study show that compared to normal (MCF10A) or hormone dependent (MCF-7) breast cells, hormone independent and resistant breast cancer cells (MCF7-2A, LCC-9, and MCF7-RR) have higher expression of the L-type and T-type calcium channels and higher concentrations of intracellular calcium. When hormone resistant (LCC-9) cells were treated with the intracellular calcium chelator BAPTA-AM, the L-type calcium channel blocker methoxyverapamil, or the T-type calcium channel blocker mibefradil, there was a significant decrease in the expression of the ERα regulated gene progesterone receptor that was reversed by the addition of estradiol. More importantly, when hormone independent (MCF7-2A) and hormone independent (LCC-9) cells were treated with the calcium channel blockers, there was in a significant decrease in growth and a re-sensitization of the antiestrogen resistant LCC-9 cells to tamoxifen and fulvestrant. Taken together, the preliminary results suggest that increased expression of calcium channels and intracellular calcium contribute to the hormone independent activation of ERα and point to a potential new therapeutic target for hormone independent and resistant breast cancer.

Citation Format: Kedra Cyrus, Mudit Kaushal, Zeina Sharawi, Glyn Noguchi, Tiffany Chang, William Rydzewski, Fatima Gibrel, William Yeguech, Bassam Haddad, Mary Beth Martin. Calcium channels: Potential new therapeutic targets for hormone independent and resistant breast 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 5040. doi:10.1158/1538-7445.AM2015-5040
Abstract 5048: The cholesterol metabolite, 27-hydroxycholesterol stimulates cell proliferation in prostate epithelial cell lines via estrogen receptor beta
Authors: Raza Shaneabbas , Schommer Jared , Meyer Megan , Guo Bin , Hammer Kimberly , Ghribi Othman
Published: August 2015

For every 6 men, 1 will be diagnosed with prostate cancer (PCa). Estrogen receptors are known to play a role in prostate carcinogenesis however, it is unclear whether the estrogenic effects are estrogen receptor α (ERα) or estrogen receptor β (ERβ) mediated. Although, it is speculated that ERα is associated with harmful effects on PCa while ERβ plays a more protective role on PCa, clinical trials using ERβ selective modulators have not shown improved clinical outcomes. Hence, the role of ERβ in PCa is yet to be understood.

Recently a cholesterol oxidized metabolite (oxysterol), 27- hydroxycholesterol (27-OHC), was found to bind to both estrogen receptors (ERs) and act as a selective ER modulator (SERM). 27-OHC is the most prevalent cholesterol metabolite present in the blood with levels increasing with oxidative stress and age, especially in men. Increased 27-OHC levels are also found in individuals with hypercholesterolemia, which is also a risk factor for PCa. The role of 27-OHC through its action on estrogen receptors in the context of PCa is yet to be determined.

We hypothesize that 27-OHC through ER activation has deleterious effect in PCa. We found that incubation with 27-OHC of RWPE-1, LNCaP and PC3 cells increased cell proliferation significantly. Using the ER inhibitor, Fulvestrant, we demonstrate that upon co-treatment with 27-OHC and fulvestrant, cell proliferation is reduced to basal levels, indicating the involvement of ERs. Interestingly, levels of ERβ, and to a lesser extent ERα, were increased following incubation of RWPE-1, LNCaP and PC3 with 27-OHC. Furthermore, in the presence of the ERβ specific inhibitor, PHTPP, we show that 27-OHC- induced proliferation is attenuated. Unexpectedly, while 27-OHC increases cell invasion in the moderately metastatic LNCaP, it decreases cell invasion in normal cells, RWPE-1 and highly metastatic, PC3.

Altogether, our results propose dysregulated levels of 27-OHC as a new risk factor for prostate cancers with low metastatic potential. This indicates a novel role for 27-OHC in the etiopathogenesis of in PCa.

Note: This abstract was not presented at the meeting.

Citation Format: Shaneabbas Raza, Jared Schommer, Megan Meyer, Bin Guo, Kimberly Hammer, Othman Ghribi. The cholesterol metabolite, 27-hydroxycholesterol stimulates cell proliferation in prostate epithelial cell lines via estrogen receptor beta. [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 5048. doi:10.1158/1538-7445.AM2015-5048
Abstract 5050: Proteome-based combined treatment strategies synergizing antagonism of androgen receptor function in prostate cancer
Authors: Lee Joo Hyoung , Mobley James A. , Ponnazhagan Selvarangan
Published: August 2015

Acquired resistance to second-generation androgen receptor (AR)-targeted therapies compels the development of novel therapeutic agents for castration-resistant prostate cancer (CRPC). We have recently reported a novel function of endostatin (ES) through direct binding and inhibition of AR in human prostate cancer (PCa) cells. Molecular characterization of the putative mechanisms of ES function on AR-positive PCa cells by global proteomic analysis revealed dramatic upregulation of CYP51A1 expression in LNCaP cells, suggesting that the increases in cholesterol biosynthesis and the resultant steroidogenesis may provide survival mechanism for PCa cells through the augmentation of androgen-AR axis. To validate this hypothesis, anti-proliferative effects of combined treatment of ES with Lovastatin or Fluconazole, inhibitor of HMG-CoA reductase or CYP5A1, were analyzed in vitro using AR-positive LNCaP cells. Results indicated a significantly enhanced ES-mediated growth inhibition on LNCaP cells by targeting both the ligand biosynthesis and receptor function. Proteomic profiling further indicated that ES significantly decreased the levels of AR, coactivator Ran (ARA24) and downregulated AR-driven expression of MCM complex 2, 3, 4 and 6, MDC1 and STMN1, suggesting that AR-targeted effects of ES include disruption of DNA replication, cell cycle propagation and cell division. We found that dual inhibition of AR nuclear transport and AR-mediated cell division by co-treatment of ES and Taxol greatly decreased PCa cell proliferation. Together, this interdisciplinary approach integrating proteomics with PCa cell function possibly suggests that antagonism of AR, which is the principal drug target for the PCa treatment, can be potentiated by the combination of ES with currently used chemotherapy agents and adjuvants. These findings further provide new insights into proteome-based approach as a useful tool to design and optimize effective therapeutic regimens for individual patients with CRPC.

Citation Format: Joo Hyoung Lee, James A. Mobley, Selvarangan Ponnazhagan. Proteome-based combined treatment strategies synergizing antagonism of androgen receptor function in 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 5050. doi:10.1158/1538-7445.AM2015-5050
Abstract 5062: Ovarian cancer cells induce the microenvironment changes and subsequently promote disease propagation and dissemination through epithelial-mesenchymal transition and enrichment of tumorigenic cells
Authors: Huser Nanni , Yan Zhengming , Yuan Jing , Zhang Cathy C.
Published: August 2015

Ovarian cancer has the highest mortality rate of all gynecological cancers primarily due to the high incidence of metastatic spread prior to diagnosis. Understanding the biology of this malignancy and developing models that reflect the patient disease phenotype will create opportunities for testing targeted therapy. We used the Ovcar 3 cell line to establish a metastasis model through serial in vivo passage. After the treatment of docetaxel and carboplatin, the characteristics of minimal residual disease were assessed. Notably, the stromal environment and tumor initiating subpopulation play critical roles on the Ovcar 3MET disease invasiveness and progression. Compared to the molecular profile of the parental cells, these invasive Ovcar 3MET cells exhibited higher expressions of β-catenin, slug, N-cadherin and ZEB1, suggesting the epithelial-mesenchymal transition in these Ovcar 3MET cells. In addition, a higher fraction of the CD44+/CD24- subpopulation was found in the Ovcar 3MET cells, suggesting enrichment of the tumorigenic population. With the treatment of chemotherapeutic agents, we observed an enrichment of CD44+/CD24- subpopulation in the minimal residual disease. The self-renewal functional analysis demonstrated a higher tumorigenic potential of the MRD cells compared to the cells from vehicle treated mice. In contrast, treatment of the dual PI3K/mTOR inhibitor PF-04691502 resulted in significant anti-tumor and anti-metastatic activities without increasing the MRD self-renewal potential. These results warrant the combination assessment of chemotherapy with targeted agents to reduce or eradicate MRD, therefore to mitigate the risk of disease relapse.

Citation Format: Nanni Huser, Zhengming Yan, Jing Yuan, Cathy C. Zhang. Ovarian cancer cells induce the microenvironment changes and subsequently promote disease propagation and dissemination through epithelial-mesenchymal transition and enrichment of tumorigenic 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 5062. doi:10.1158/1538-7445.AM2015-5062