bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 522: Role and regulation of Aurora A in radiation resistance mechanisms in glioblastoma
Authors: Ramasubramanian Brinda , Palanichamy Kamalakannan , Patel Disha , Haque Saikh Jaharul , Chakravarti Arnab
Published: July 2016

Glioblastoma (GBM) is the most aggressive tumor of the human brain, which inevitably escapes the standard treatment modalities that include surgery, radiotherapy and chemotherapy. Consequently, the median survival of GBM patients remains 12-15 months despite these aggressive treatments. Therefore, to develop effective therapies, it is important to understand how GBM cells develop therapeutic resistance. Aurora A is a ubiquitously expressed serine/threonine kinase, which plays essential roles in mitotic regulation. This protein kinase, which phosphorylates p53, PP1, CEBP, and histone H3 among other regulators of cell division, is overexpressed in various neoplasms including GBM and invariably associated with poor prognosis. Recent studies have demonstrated that Aurora A contributes to radio-resistance in various solid neoplasm including GBM. Importantly, Aurora A inhibitors are currently in phase I/II clinical trials for solid cancer. Notably, Aurora A inhibitor MLN8237 that crosses the blood brain barrier specifically inhibits Aurora A at concentrations ≤ maximally tolerated dose in animal models and in phase I clinical trials. We found that Aurora A protein level is upregulated by ionizing radiation in GBM cells. This was, in part, mediated by radiation-induced inhibition of 26S-proteosomal degradation of Aurora A. Aurora A was silenced using both lentiviral mediated shRNA expression and a specific Aurora A inhibitor in three different established cell lines, and were treated with 6 gy radiation. We found that pharmacological inhibition and RNAi-mediated knockdown of Aurora A sensitized the GBM cells to radiation therapy resulting in reduced cell proliferation and increased apoptosis. Interestingly, we observed that RNAi-mediated down-regulation of Aurora A was associated with a decrease in X-linked inhibitor of apoptosis (XIAP) expression in GBM cells. We also found that Aurora A and XIAP were co-localized in GBM cells. Taken, together our results reveal a mechanism by which Aurora A confers radio-resistance in GBM cells and further rationalize the use of Aurora A inhibitors as radio-sensitizer in solid neoplasms including GBM.

Funding sources: This work was supported by R01CA108633 (To AC), 1RC2CA148190 (To AC) U10CA180850-01 (To AC), 1R01CA169368 (To AC) from the National Cancer Institute (NCI), Brain Tumor Funders Collaborative Grant (To AC), Ohio State University Comprehensive Cancer Center Award (To AC)

Citation Format: Brinda Ramasubramanian, Kamalakannan Palanichamy, Disha Patel, Saikh Jaharul Haque, Arnab Chakravarti. Role and regulation of Aurora A in radiation resistance mechanisms in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 522.
Abstract 5235: Dietary administration of δ- and γ-tocopherol inhibits estrogen-mediated mammary tumorigenesis in ACI rats
Authors: Das Gupta Soumyasri , Wahler Joseph , Bak Min Ji , Lee Mao-Jung , Lin Yong , Joe Shih Weichung , Yang Chung S , Suh Nanjoo
Published: July 2016

Tocopherols (T), members of the vitamin E family, occur in four forms designated as α, β, δ and γ. Epidemiological studies suggest that tocopherols could reduce the risk of cancer. α-T is known as the classical vitamin E, being the predominant form of tocopherol found in human blood and tissues. However, clinical studies with α-T to substantiate the role of tocopherols in cancer prevention have provided inconclusive results. Such outcomes indicate that the cancer preventive activity of tocopherols might be attributed to the other forms of tocopherols, but not α-T. γ-T, which is the most abundant tocopherol in vegetable oils, is our major dietary source of tocopherols. Recent studies have demonstrated that δ-T and γ-T have higher anti-cancer activity than α-T in several animal models of cancer. The present study investigated the cancer preventive activity of individual purified forms of tocopherols α-T, δ-T, γ-T as well as a naturally occurring γ-T rich mixture of tocopherols (γ-TmT) in an in vivo model of estrogen receptor (ER)-positive breast cancer, namely estrogen supplemented August-Copenhagen Irish (ACI) rats. ACI rats were subcutaneously implanted with 9 mg of 17β-estradiol (E2) in silastic tubings and administered with diets containing 0.2% α-T, δ-T, γ-T or γ-TmT. Rats were euthanized at 30 weeks of mammary carcinogenesis when the E2 control group exhibited 100% tumor incidence. α-T had no effect on estrogen-induced mammary tumors. However, treatment with 0.2% δ-T, γ-T and γ-TmT reduced the tumor volume by 51% (p<0.05), 60% (p<0.01) and 59% (p<0.01), respectively. Administration of α-T, δ-T and γ-T resulted in a 2.1-, 80- and 62-fold increase of these tocopherols, respectively, in the serum of

ACI rats. Supplementation with the individual tocopherols also increased the levels of their respective short chain metabolite, carboxyethyl hydroxychromans (CEHCs) in the serum. Administration of δ-T and γ-T was associated with 185- and 99-fold increase in their CEHC metabolites, respectively. The high serum levels of δ- and γ-CEHC metabolites could contribute to the enhanced chemopreventive activity of δ-T and γ-T. In conclusion, δ-T, γ-T and γ-TmT could be potential natural agents for the prevention of estrogen-induced mammary tumorigenesis.

(This work was supported by the NIH grant R01 AT007036 and the New Jersey Commission on Cancer Research Postdoctoral Fellowship to Soumyasri Das Gupta).

Citation Format: Soumyasri Das Gupta, Joseph Wahler, Min Ji Bak, Mao-Jung Lee, Yong Lin, Weichung Joe Shih, Chung S Yang, Nanjoo Suh. Dietary administration of δ- and γ-tocopherol inhibits estrogen-mediated mammary tumorigenesis in ACI rats. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5235.
Abstract 564: Combination therapy of chemokine receptor inhibition plus PD-L1 blockade potentiates antitumor effects in a murine model of breast cancer
Authors: Jung Heiyoun , Ertl Linda , Ebsworth Karen , Janson Christine , Zhang Penglie , Sreenivas Punna , Schall Thomas , Charo Israel
Published: July 2016

Trafficking and expansion of myeloid derived suppressor cells (MDSCs) plays a major role in the immune suppression of tumors. MDSCs express chemokine receptors which likely mediate their recruitment to the tumor microenvironment. Suppression of T cells is also mediated by the interaction of programmed death-1 (PD-1) and its ligands which are abundantly expressed in cancer cells and immune infiltrates, including MDSCs. Here, we show that targeting both pathways through administration of a small molecule chemokine receptor antagonist (CCX9588, which blocks CCR1) and a PD-L1 monoclonal antibody (mAb) significantly reduced tumor burden in mice who received orthotopic transplants of 4T1 cells, a cell line used to model triple negative breast cancer. Primary tumor growth was modestly reduced by either agent (PD-L1 mAb or CCR1 inhibitor) alone, but the combination of CCR1 inhibitor plus PD-L1 mAb resulted in significantly reduced tumor progression as compared to either of the single agents. Furthermore, lung metastasis was also significantly reduced by CCR1 antagonist and combination treatment. Orthotropic 4T1 cell engraftment induced robust expansion of CD11b+Ly6Ghi Ly6Chi MDSCs, a subpopulation of which express CCR1. Analysis of the tumor infiltrating cells revealed that CCX9588 significantly reduced the number of MDSCs and increased CD8 T cells infiltration in primary tumors, suggesting that CCR1 blockade of MDSC trafficking in combination with PD-L1 mAb translates into reduced tumor burden, possibly through increased CD8 T cell response against the tumor. Analysis of human breast cancer patient samples from The Cancer Genome Atlas (TCGA) database revealed that the CCR1 ligands, MCP-7 (CCL7) and RANTES (CCL5) are present at significantly higher levels in breast cancers as compared to normal breast tissue. Interestingly, ligands for CCR1 and PD-1 are significantly higher in triple negative breast cancer samples than in the other breast cancer subtypes. These data are the first to show that CCR1 chemokine receptor antagonists can act synergistically with PDL-1 inhibitors, and suggest a novel approach for potentiating the activity of immune cell checkpoint inhibitors in one of the most aggressive forms of breast cancer.

Citation Format: Heiyoun Jung, Linda Ertl, Karen Ebsworth, Christine Janson, Penglie Zhang, Punna Sreenivas, Thomas Schall, Israel Charo. Combination therapy of chemokine receptor inhibition plus PD-L1 blockade potentiates antitumor effects in a murine model of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 564.
Abstract 604: A multi-layered, hydrogel system for automated 3D high throughput drug screening of cancer-stromal cell co-cultures
Authors: Constantinou Pamela E. , Engel Brian J. , Sablatura Lindsey K. , Doty Nathaniel J. , Carson Daniel D. , Farach-Carson Mary C. , Harrington Daniel A. , Zarembinski Thomas I.
Published: July 2016

Pre-clinical drug screens, involving culturing well-annotated cancer cell lines on two-dimensional (2D) tissue culture plastic, poorly recapitulate in vivo tumor characteristics and yield candidate drugs which fail clinical trials. We created a multi-layer, hyaluronic acid (HA)-based hydrogel system that incorporates three layers: an acellular cushion layer; an encapsulated cancer cell layer for growth in three dimensions (3D); and a collagen-containing layer that supports the growth of stromal cells on top of the hydrogel (2.5D). Utilizing high-throughput robotic delivery coupled with automated advanced imaging, this formulation provides a highly reproducible system for spheroid culture of prostate (C4-2B) or endometrial (Ishikawa) cancer cell lines in mono- or co-culture with matched stromal cells (HS27a or ESS-1, respectively). Both culture systems provided high cancer cell viability over one week of culture. Cells were treated with drugs from a panel of chemotherapeutic compounds. Cells cultured in our 3D multi-layer HA-based system responded to cytotoxic drugs distinctly from cells grown in 2D and 3D-aliginate, and better predicted the efficacy of chemotherapeutics in clinical trials. We have also successfully incorporated cells derived from patient derived xenograft (PDX) models in our 3D culture system. Wider adoption of 3D automated screening has the potential to speed drug discovery and increase success of drugs in clinical trials.

Citation Format: Pamela E. Constantinou, Brian J. Engel, Lindsey K. Sablatura, Nathaniel J. Doty, Daniel D. Carson, Mary C. Farach-Carson, Daniel A. Harrington, Thomas I. Zarembinski. A multi-layered, hydrogel system for automated 3D high throughput drug screening of cancer-stromal cell co-cultures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 604.
Abstract 605: Building better cell models for screening oncology compounds
Authors: Evans David M. , Selby Michael , Delosh Rene , Laudeman Julie , Ogle Chad , Reinhart Russell , Silvers Thomas , Parchment Ralph E. , Teicher Beverly
Published: July 2016

The NCI60 screen is a valuable compound screening service offered to researchers for 20+ years. The screen allows the submission of small molecules, biologics and natural products for testing in the NCI60 cell line panel (60 cell lines covering 9 major tumor types). Sophisticated tools such as COMPARE allow stratification of compounds based on pattern of cell line response and these tools elucidated several compounds with differential specificity for a disease type (e.g. breast cancer over melanoma).

Since inception the NCI60 screen has been performed in 96-well plates on monolayer cultures of cells. SulfoRhodamine B was used to measure cell viability after a 48h exposure to the compounds at either a single concentration or 5 point concentration response assay. However, recent work has suggested that cells in 3D culture may respond differently to compounds than in 2D. 3D culture systems produce tumor cell spheroids that exhibit features seen in vivo including greater cell-cell contact, and gradients of nutrients and oxygen between the periphery and the center of the spheroid. We sought to optimize the NCI60 cells for use in a 3D spheroid model that would be suitable for screening compounds. By varying NCI60 cell plating density and incubation times, we optimized the formation of spheroids of a given diameter (500um) in Ultra Low Attachment (ULA) plates. Spheroids were imaged using a high content imaging plate reader and classified into 4 categories based on their apparent morphologies. Comparisons were made in drug sensitivity between 2D and 3D model systems using the NCI60 and cells derived from PDX samples. Data on the optimal cell densities of the NCI60 lines used for spheroid formation as well as concentration response effects of select agents compared in 2D and 3D models are presented. These data will allow others to rapidly utilize these 3D models to determine the differential drug sensitivity of select cell types. Funded by NCI Contract No. HHSN261200800001E.

Citation Format: David M. Evans, Michael Selby, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Thomas Silvers, Ralph E. Parchment, Beverly Teicher. Building better cell models for screening oncology compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 605.
Abstract 618: Dual PI3K/mTOR inhibition as a treatment strategy for PIK3CA and APC mutant colorectal cancers
Authors: Payne Susan , Foley Tyler , Pasch Cheri , Yueh Alexander , Korkos Demetra , Van De Hey Dana , Clipson Linda , Deming Dustin
Published: July 2016

BACKGROUND: Colorectal cancer (CRC) is understood as a diverse group of cancers calling for the personalization of therapeutics to individual subtypes of CRC. Mutations in PIK3CA, which result in a constitutively active phosphoinositide-3-kinase protein (PI3K), are present in 20-30% of CRCs. Aberrations in the Adenomatous Polyposis Coli (APC) gene are found in 86% of PIK3CA mutant CRCs. The potential for treatment of tumors with this specific profile is of great interest. METHODS: Mice expressing a constitutively active PI3K and loss of APC were generated and spheroid cultures were derived from the resulting cancers. PIK3CA and APC mutant spheroids were treated with GDC0941 (GDC), a pan PI3K inhibitor, NVP-BYL719 (BYL), a PI3K alpha isomer specific inhibitor, or NVP-BEZ235 (BEZ), a dual mTOR/PI3K inhibitor. Proliferation was measured by changes in spheroid diameter over time. BEZ treatment of mice with PIK3CA and APC mutant CRCs was performed over 14 days. Tumor response was measured with serial murine colonoscopy and dual hybrid 18F-FDG PET/CT imaging. RESULTS: Persistent PIK3CA and APC mutant CRC spheroid growth was observed with BYL and GDC treatment, while BEZ treatment resulted in a significant reduction in spheroid size. Immunoblotting determined that this correlated with significant suppression of phosphorylated AKT, RPS6 and 4EBP1 in BEZ-treated spheroids and incomplete PI3K pathway inhibition following BYL and GDC treatment. BEZ treatment was then investigated in mice with PIK3CA and APC mutant CRCs. BEZ elicited a dramatic treatment response in these cancers on endoscopy, with a 53% decrease in median lumen occlusion compared to a 60% increase in controls over the 14 day treatment period. PET/CT imaging results confirmed these findings demonstrating a decrease in tumor size and avidity post BEZ treatment. BEZ resulted in inhibition of the PI3K pathway in these tumors. Reactivation of PI3K signaling was observed within 24 hours of the withdrawal of BEZ. BEZ was well-tolerated in these mice and associated with a decrease in the endoscopic anemia score. CONCLUSIONS: PIK3CA and APC mutant CRCs are a potentially targetable subtype of cancer. These cancers are resistant to proximal inhibition of the PI3K pathway, but are responsive to dual PI3K/mTOR inhibition. These results warrant further investigation in clinical studies.

Citation Format: Susan Payne, Tyler Foley, Cheri Pasch, Alexander Yueh, Demetra Korkos, Dana Van De Hey, Linda Clipson, Dustin Deming. Dual PI3K/mTOR inhibition as a treatment strategy for PIK3CA and APC mutant colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 618.
Abstract 626: Application of patient-derived xenograft model in nasopharyngeal carcinoma research
Authors: Hsu Cheng-Lung , Kuo Yung-Chia , Huang Yenlin , Huang Yin-Cheng , Lui Kar-Wai , Chang Kai-Ping , Lin Tung-Liang , Fan Hsien-Chi , Lin An-Chi , Hsieh Chia-Hsun , Lee Li-Yu , Wang Hung-Ming , Li Hsin-Pai , Chao Angel , Chang Yu-Sun
Published: July 2016

Nasopharyngeal carcinoma (NPC) was an Epstein Barr virus (EBV)-related malignancy and tumor microenvironment had a pivotal role in tumor progression. Paucity of good NPC animal models hindered the research in this field. Recently, patient-derived xenograft (PDX) had been shown to be a good preclinical model for drug screening and cancer related research. We had developed two PDX mice lines from engrafting NPC metastatic tumors. Positive EBV-encoded small RNAs staining confirmed these tumors harboring EBV. Further gene expression profile analysis showed higher similarity of PDX to primary parent tumor than NPC cell line xenograft. In vivo drug screening in the PDX system demonstrated gemcitabine had the best antitumor effect among the tested drugs. In this PDX corresponding patient also showed excellent response to gemcitabine treatment. Combination of gemcitabine and valproic acid had synergistic antitumor effect. Further adding ganciclovir in this two combined regimen enhancing cytolytic viral activation had the best antitumor response among the tested regimens. This three combined regimen treated group had lower plasma EBV-DNA load and tumor viral concentration and less viable tumor cells than gemcitabine + valproic acid group. These promising results would open a new era for EBV-targeting therapy in NPC treatment.

Citation Format: Cheng-Lung Hsu, Yung-Chia Kuo, Yenlin Huang, Yin-Cheng Huang, Kar-Wai Lui, Kai-Ping Chang, Tung-Liang Lin, Hsien-Chi Fan, An-Chi Lin, Chia-Hsun Hsieh, Li-Yu Lee, Hung-Ming Wang, Hsin-Pai Li, Angel Chao, Yu-Sun Chang. Application of patient-derived xenograft model in nasopharyngeal carcinoma research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 626.
Abstract 64: Identification and validation of novel therapeutic targets of osteosarcoma using cutting edge technologies
Authors: Smeester Branden , Wolf Natalie , Moriarity Branden S.
Published: July 2016

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. The current treatment options have not changed over the last four decades and rely on tumor resection and nonspecific combination chemotherapy, resulting in a 5-year survival rate of 0-29% if clinically apparent metastases are present. Although these tumors are rare in humans, with ∼900 cases annually, they are highly prevalent in canines, with over 50,000 cases annually, and have seemingly similar genetics. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS- associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR, MAPK, and ERK5 signaling pathways. We identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma. We have now begun pre-clinical testing of inhibitors of our top actionable targets to treat osteosarcoma, namely SEMA4D and CSF1R. Further, we have begun to validate top candidate metastasis genes using the CRISPR nuclease system in immortalized osteoblasts and osteosarcoma cell lines. Preliminarily we have found that loss of candidate osteosarcoma metastasis genes ANAPC1 or TOAK1 increases the migration capacity of immortalized osteoblasts, while loss of EXOC1 or GRLF increases their ability for anchorage independent growth in soft agar assay. Results of these ongoing studies will be presented.

Citation Format: Branden Smeester, Natalie Wolf, Branden S. Moriarity. Identification and validation of novel therapeutic targets of osteosarcoma using cutting edge technologies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 64.
Abstract 650: An isogenic experimental model identifies β-catenin as a molecular target in ovarian cancer metastasis
Authors: To Sally KY , Wong. Alice ST
Published: July 2016

Ovarian cancer metastasis is closely associated with unfavorable outcomes, yet the underlying mechanisms remain obscure. Here we establish an isogenic model that could mimic the spontaneous metastasis of human ovarian cancer. Given that tumor cells are heterogeneous in nature, an isogenic pair of highly metastatic (HM) and non-metastatic (NM) cell lines with opposite metastatic capabilities was derived using in vitro and in vivo selection. Incorporation of the luciferase gene into the cell pair allowed non-invasive monitoring of the metastatic events by bioluminescent imaging in vivo. Orthotopic implantation of HM into the ovarian bursa of NOD/SCID mice resulted in metastases within the peritoneum with ascites formation, thus representing the major dissemination pattern of human ovarian cancer cells. However, NM failed to form detectable metastases, although it was tumorigenic at the ovarian bursa. In comparison with NM, HM displayed higher spheroid-forming ability and had higher expression of stemness marker genes, which are characteristics of cancer stem-like population. By proteomic profiling and pathway analysis, HM is found to be enriched in the oncogenic β-catenin signaling, a pathway elevated in ovarian cancer metastases. Tumor initiation and metastasis of HM was dramatically impaired when β-catenin was specifically knocked down. We further demonstrated that β-catenin could down-regulate the expression of Dicer, a major component of the microRNA machinery. These results together suggest that this model could help to delineate the molecular mechanisms for ovarian cancer metastasis, and provide clinically relevant insights to target metastasizing ovarian cancer cells.

Citation Format: Sally KY To, Alice ST Wong. An isogenic experimental model identifies β-catenin as a molecular target in ovarian cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 650.
Abstract 666: Apc restoration promotes cellular differentiation and reestablishes crypt homeostasis in colorectal cancer
Authors: O’Rourke Kevin P. , Dow Lukas E. , Lowe Scott W.
Published: July 2016

Colorectal cancer (CRC) accounts for almost 10% of all cancer mortality in the United States. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. Using a novel mouse model in which Apc can be conditionally suppressed using a doxycycline-regulated shRNA, we previously showed that Apc suppression in adenomas and carcinomas drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. However, this study was unable to explore Apc action in metastasis, which is the most common cause of death in CRC patients, since models do not live long enough for metastatic dissemination to occur. To produce a system that would enable further disease progression, we generated a comprehensive panel of colon organoid cultures that reflect iterative combinations of the 3 most common genetic lesions in human CRC: Apc-mut, Apc-mut;KrasG12D, Apc-mut;p53-mut and Apc-mut;KrasG12D;p53-mut. These cultures can be transplanted orthotopically into the colon of recipient mice or seeded in the circulation to assess primary tumor behavior and metastatic dissemination. We show that Apc silencing in organoid culture results in the dysregulated proliferation of stem and progenitor cells but this is reversible upon Apc restoration or pharmacologic inhibition of the Wnt pathway. When engrafted into the colon of recipient mice, these cells create ∼1-3 focal colon tumors that can be tracked using colonoscopy, ultrasound and bioluminescence imaging, and are largely indistinguishable from tumors that develop in transgenic models. Most importantly, triple mutant (Apc/Kras/p53) organoids form invasive adenocarcinoma and colonize the lung following tail vein injection, providing a setting to dissect the importance of Wnt pathway activity in various stages of disease progression. Ultimately, the ability to study cellular and molecular phenotypes in vitro, and assess tumor development in vivo, provides a flexible system to explore the genetic factors underlying CRC progression and metastasis, and overcomes many of the previous technical challenges of modelling advanced CRC.

Citation Format: Kevin P. O’Rourke, Lukas E. Dow, Scott W. Lowe. Apc restoration promotes cellular differentiation and reestablishes crypt homeostasis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 666.
Abstract 668: Induction of mammary carcinoma in tetracycline-inducible cMET(T1010I) transgenic mice
Authors: Yang Chuanwei , Gonzalez Ana M. , Ueno Naoto T. , Liu Shuying , Zhang Jinsong , Mills Gordon B. , Mendelsohn John , Ram Prahlad
Published: July 2016

Gene amplification and mutations of cMET have been reported in a wide variety of tumors, including colorectal, lung, renal, liver, thyroid, and breast cancer. cMET(T1010I) mutation occurred in 5.2% out of 148 sibling pairs diagnosed with colorectal cancer in one study. This germline mutation occurs in about 1% of breast cancer patients, but its frequency increases to 2% in metastatic breast cancer. Furthermore, 16% of breast cancer cases with PI3KCA mutations have co-mutations of the cMET oncogene. To determine the functional consequences of this germline mutation and elucidate the molecular mechanisms of tumorigenesis induced by its oncogenic mutant protein, we developed a transgenic mouse model expressing human cMET(T1010I) under the control of a Tet-responsive promoter. MMTV promoter - directed expression of reverse tetracycline-controlled transactivator (rtTA) turns on the expression of the cMET(T1010I) transgene in murine mammary glands upon tetracycline administration. Thirty percent of these mice developed early onset mammary carcinoma within 5 months of transgene induction. This contrasts with previous reports of mammary tumor development in MMTV-cMET transgenic mice after more than a year. This strong phenotype is not the result of a random insertional effect of the transgene as no mammary tumors developed in the 29 transgene positive mice without mammary-specific expression of rtTA over a year in our study. Molecular characterization of these tumors is currently underway and the results may reveal mechanisms of tumorigenesis and novel therapeutic targets for patients harboring this mutation. In conclusion, we showed for the first time that the cMET(T1010I) mutation can trigger rapid breast tumorigenicity. By using organ-specific expression of rtTA to direct the expression of cMET(T1010I) in the organ of interest, our transgenic model may be used to understand the impact of cMET(T1010I) on many other cancer types such as colorectal, lung, renal, thyroid, and liver cancer.

Citation Format: Chuanwei Yang, Ana M. Gonzalez, Naoto T. Ueno, Shuying Liu, Jinsong Zhang, Gordon B. Mills, John Mendelsohn, Prahlad Ram. Induction of mammary carcinoma in tetracycline-inducible cMET(T1010I) transgenic mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 668.
Abstract 684: Targeting an unconventional kinase-invasion axis in breast cancer metastasis
Authors: Matossian Margarite D. , Elliott Steven , Hoang Van T. , Burks Hope E. , Phamudy Theresa B. , Chrisey Doug , Zuercher William J. , Drewry David H. , Wells Carrow , Collins-Burow Bridgette , Burow Matthew E.
Published: July 2016

Triple negative breast cancer (TNBC) is associated with poor survival, metastatic recurrence, increased mortality, and has few viable therapeutic options. Given the high mortality associated with metastasis of this disease, discovery of therapeutics targeting the metastasis axis is imperative. Kinases have been demonstrated to have an essential role in regulation of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Elucidating specific signaling pathways that promote EMT has the potential to provide novel targeting strategies in the treatment of TNBC. Here we demonstrate a partially non-selective polo-like kinase 1 (PLK1) inhibitor, GSK346294, is capable of EMT reversal in phenotypically mesenchymal TNBC cell lines, as evidenced by enhanced E-cadherin expression and loss of cellular migration potential. Structurally analogous but highly specific PLK inhibitors did not exhibit similar EMT changes, which led us to investigate off-target kinases inhibited by GSK346294. Follow-up kinase profiling studies revealed members of the NEver-in-mitosis-related Kinase family (NEK5 and NEK9) as additional kinases inhibited by GSK346294, leading us to investigate their individual roles in the progression of EMT. NEK5 overexpression in a non-invasive breast cancer cell line increased both migration and invasion in trans-well assays. Additionally, enhanced NEK5 expression altered the TNBC gene expression profile, enhancing expression of genes associated with growth factor signaling (MET, EGFR) and the mesenchymal gene signature (SLUG, VIM). To date, no studies exist which delineate the regulatory roles of NEK5 and NEK9 in EMT or cellular invasion/motility. Further investigation into the function of NEK5 and/or NEK9 in the metastatic progression may provide novel therapeutic targets for the treatment of TNBC, the implication of which could impact the treatment and management of neoplastic disease and metastasis beyond breast cancer.

Citation Format: Margarite D. Matossian, Steven Elliott, Van T. Hoang, Hope E. Burks, Theresa B. Phamudy, Doug Chrisey, William J. Zuercher, David H. Drewry, Carrow Wells, Bridgette Collins-Burow, Matthew E. Burow. Targeting an unconventional kinase-invasion axis in breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 684.
Abstract 696: Targeting the proprotein convertase PCSK6/PAECE4 abrogates human melanoma malignant phenotype
Authors: Siegfried Geraldine , Imbard Apolline , Evrard Serge , Khatib Abdel-Majid
Published: July 2016

The proprotein convertases (PCSKs) are involved in the proteolytic maturation/activation of a wide range of protein precursors involved in neoplasia such as adhesion molecules, growth factors, growth factor receptors, and metalloproteinases. Expression analysis of all the PCSKs family members, namely PC1, PC2, furin, PC4, PC5, PACE4, and PC7 in various human and murine melanoma cells revealed increased PCSK6/PAECE4 expression while compared to melanocytes. The use of in vitro digestion assays and cell transfection experiments revealed that targeting the PCSK6/PAECE4 in melanoma cells using small interfering RNA (siRNA) reduced PCSKs activity and repressed the processing the PCSKs substrates proIGF-1R, pro-VEGF-C and proPDGF-A. Theses unprocessed substrates failed to mediate their signaling pathways that associated reduced cell proliferation. Furthermore, PCSK6/PAECE4 gene silencing reduced melanoma cells migration and invasion that paralleled decreased gelatinase MMP-2 and MMP-9 activity and altered expression and secretion of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2, urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Taken together, these findings highlight the importance of PCSK6/PAECE4 activity in melanoma cells and suggest PCSK6/PAECE4 inhibition as a potentially promising strategy for the prevention of melanoma invasiveness.

Citation Format: Geraldine Siegfried, Apolline Imbard, Serge Evrard, Abdel-Majid Khatib. Targeting the proprotein convertase PCSK6/PAECE4 abrogates human melanoma malignant phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 696.
Abstract 7: LKB1 deficiency is associated with a unique metabolic signature in Kras mutant non-small cell lung cancer (NSCLC)
Authors: Liu Tingyu , Sennott Erin , Zhong Zhengjie , Steadman Mya , Marsh Kelly , Hurov Jonathan , Benes Cyril , Engelman Jeffrey A.
Published: July 2016

LKB1 deficiency is found in approximately 10% of non-small cell lung cancer harboring Kras mutation. Loss of LKB1 has been associated with increased metastatic rates and decreased survival in patients. Currently, no effective therapy has been developed against this subtype of lung cancer. LKB1 can regulate multiple cellular activities. One of the most well-studied functions is its regulation on cell metabolism through AMPK signaling pathway. How LKB1 loss globally impacts NSCLC metabolism has not been well understood yet. Using isotope-labeled substrate tracing approach, we found an increase of specially-labeled intermediates in pentose phosphate pathway (PPP) that may suggest upregulation of PPP activity. In addition, we also performed an unbiased drug screen that showed higher sensitivity to an mTOR inhibitor in Kras and LKB1 double mutant lung cancer cells relative to cancer cells under other genetic background. These data suggest that loss of LKB1 may upregulate biosynthetic reactions in Kras mutant NSCLC and render these cells sensitive to inhibitors in biosynthesis. These findings may provide new insights in developing therapeutic agents targeting KrasMT/LKB1MT NSCLC.

Citation Format: Tingyu Liu, Erin Sennott, Zhengjie Zhong, Mya Steadman, Kelly Marsh, Jonathan Hurov, Cyril Benes, Jeffrey A. Engelman. LKB1 deficiency is associated with a unique metabolic signature in Kras mutant non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 7.
Abstract 701: Loss of Krupple-like factor 10 enhanced epithelial-mesenchymal transition of pancreatic cancer by modulating glucose metabolism via Sirt 6 and PKM2
Authors: Tsai Yi-Chih , Chen Su-Liang , Chang HS Vincent , Ch’ang. Hui-Ju
Published: July 2016

Introduction:

Pancreatic Cancer is well known for its deregulated TGFâ signaling. We have reported that the expression level of Krüpple like factor 10 (Klf10), a TGFâ early response transcription factor, is associated with clinical outcomes of pancreatic cancer patients. In this study, we would like to evaluate the mechanisms of Klf10 in promoting distant metastasis of pancreatic cancer.

Materials and Methods:

Klf10 over-expressing- or depleting-human pancreatic cancer cell lines were established. Western blot was used to evaluate epithelial-mesenchymal transition (EMT) markers and glycolysis related molecules. Cancer cell migration was measured by trans-well assay. Luciferase-labeled Panc-1 cells with or without Klf10 mRNA silencing were injected via tail veins of NOD/SCID mice to evaluate their metastatic ability. The glucose consumption, lactate production and mitochondrial respiration of pancreatic cancer cells were measured by assay kit or XFe Extracellular Flux Analyzers.

Results:

Down-regulating Klf10 in Panc-1 cells was associated with enhanced migration and invasion ability by in vitro and in vivo studies. Immunoblots of cell lysates revealed decreased expression level of E-cadherin and increased Twist, MMP9. Glycolysis activity was elevated while mitochondrial respiration decreased after silencing Klf10 mRNA in Panc-1 cells. The phenomenon was in parallel with decreased Sirt 6 and increased HIF-1á as well as PKM2 expression. We demonstrated that Klf10 bind to the promoter and transcriptionally regulated Sirt 6. Modulating Sirt 6 and PKM2 reversed the EMT and metastatic phenotype of Panc-1 cells with Klf10mRNA silencing.

Discussion:

Loss of Klf10 enhanced the migratory ability of pancreatic cancer cells. Klf10 transcriptionally regulated Sirt 6 which modulated glycolytic enzymes including PKM2 via HIF-1á. Elevated PKM2 promoted EMT phenotype and distant metastasis of pancreatic cancer. Klf10 is a potential prognostic and therapeutic biomarker for pancreatic cancer.

Citation Format: Yi-Chih Tsai, Su-Liang Chen, HS Vincent Chang, Hui-Ju Ch’ang. Loss of Krupple-like factor 10 enhanced epithelial-mesenchymal transition of pancreatic cancer by modulating glucose metabolism via Sirt 6 and PKM2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 701.
Abstract 707: High expression of Pol é promotes invasion and metastases in esophageal squamous cell carcinoma
Authors: Zou Shitao , Wu Jinchang , Ding Wei-Qun , Zhou Jundong
Published: July 2016

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DNA polymerase iota (Pol iota) is an error-prone DNA polymerase involved in translesion DNA synthesis (TLS) that may play a significant role in the accumulation of DNA mutations. Our previous studies identified the elevated expression of Pol iota in human esophageal squamous cell cancer (ESCC) tissues and revealed that Pol iota contributes to ESCCs’ progression. The present study aimed to investigate the molecular mechanism by which Pol iota enhances the invasiveness and metastasis of ESCC cells. We found that the expression of Pol iota was higher in ESCCs with lymph node metastasis compared to those without lymph node metastasis. Kaplan-Meier analysis revealed a negative correlation between the expression of Pol iota and patients’ prognosis. Furthermore, the expression of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9), both being essential regulators for cells invasion, were associated with that of Pol iota in ESCCs tissue samples. The wound healing and transwells assay revealed that over-expression of Pol iota enhances motility and invasiveness of ESCC cells. In vivo, up-regulation of Pol iota promoted the colonization of ESCC cells in the liver, lung and kidney. Signaling pathway analysis showed that Pol iota induces the expression of MMP-2/9 and enhances the ESCCs progression via JNK-AP-1 cascade. Altogether, our finding demonstrated the underlying mechanism by which Pol iota promotes ESCCs’ development.

Citation Format: Shitao Zou, Jinchang Wu, Wei-Qun Ding, Jundong Zhou. High expression of Pol é promotes invasion and metastases in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 707.
Abstract 71: Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair
Authors: Gutschner Tony , Hämmerle Monika , Genovese Giannicola , Draetta Giulio F. , Chin Lynda
Published: July 2016

The comprehensive and coordinated efforts of The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) identified the somatic genomic alterations occurring in a broad panel of human cancers. The challenge ahead is to understand the functions of these somatically altered genes.Genome editing tools like the CRISPR/Cas9 system represent a transformative technology that allows the precise engineering of cells harboring specific (point) mutations found in specific tumor subtypes, enabling the investigation of the role of a candidate cancer gene in a stringently defined genetic context. Once recruited to its DNA target sequence in cells, Cas9 induces a DNA double strand break (DSB), which is either repaired by the non-homologous end-joining pathway (NHEJ), or by homology-directed repair (HDR). The HDR pathway allows for a precise site-specific knock-in and thus functional analysis of cancer-associated sequence variants. However, HDR activity is restricted to the late S and G2 phases of the cell cycle, while the NHEJ pathway dominates DNA repair throughout the cell cycle. Thus, the error-prone NHEJ pathway out-competes the HDR pathway, which reduces the likelihood for precise insertions, deletions or base substitutions at the DSB.

Here, we devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1 resulting in a cell cycle tailored expression with low levels in G1, but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the relative rate of HDR by up to 87% at a single-copy reporter locus, and up to 42% at the endogenous MALAT1 locus compared to wild-type Cas9.

In summary, our regulatory concept takes advantage of cell-autonomous pathways to enable fluctuation of Cas9 protein levels coupled to cell cycle dynamics. This resulted in higher site-specific integration events without sophisticated manipulation of cells by small molecules or additional gene targeting reagents. Future methodological developments may enable high-resolution expression of Cas9 and alternative genome engineering proteins like Cpf1 by combining cell cycle phase-specific transcriptional and post-translational regulatory elements. This might enhance HDR efficiencies even further, and will help to make sense of cancer genomic data.

Citation Format: Tony Gutschner, Monika Hämmerle, Giannicola Genovese, Giulio F. Draetta, Lynda Chin. Cas9 protein engineering for cell cycle-specific genome editing to enhance homology directed repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 71.
Abstract 716: STAT1 expression in the tumor-stroma microenvironment is influenced by loss of PTEN in prostate cancer
Authors: Vidotto Thiago , Picanço-Albuquerque Clarissa G. , Saggioro Fabiano P. , Borges dos Reis Rodolfo , Siemens David Robert , Koti Madhuri , Squire Jeremy A.
Published: July 2016

The prostate cancer (PCa) microenvironment is an admixture of stromal cells, such as lymphocytes, fibroblasts, myofibroblasts and blood vessel endothelium. The reactive stromal microenvironment of PCa facilitates tumor growth through cytokine and chemokine-mediated cell signaling. STAT1, a transcription factor, induces cell cycle arrest, apoptosis and modulates immune and inflammatory response through IFN-á/ã cytokine activation. IFN-á/ã may be secreted in response to PI3K/AKT activation, since this pathway is associated with JAK/STAT signaling. In addition, the PI3K/AKT pathway can become activated when the PTEN prognostic biomarker is deleted in PCa. The rationale for this project is that the immediate stromal microenvironment of PCa may become pro-tumorigenic as a result of induction of inflammatory cytokines caused by PTEN deletions and activation of PI3K/AKT. We performed FISH analysis of the PTEN gene on 172 cores on a PCa tissue microarray (TMA) derived from 43 patients with intermediate risk disease. Analysis of cytoplasmic and nuclear STAT1 in both the tumor and stromal compartments was performed on each TMA core using immunohistochemistry (IHC) to determine whether the tumor PTEN deletion status was associated with STAT1 expression. In each core, tumor and stromal cell histologies were evaluated independently to estimate the percentage area that was stained positive by IHC. The staining was graded as percentage of positive cells with 0 (negative), 1 (less than 10%), 2 (11% to 50%), and 3 (more than 51%). We found that STAT1 expression in stroma was increased when the PTEN gene in the adjacent tumor was homozygously deleted (p = 0,04). We also showed that mean STAT1 expression in benign tissue cores was reduced when compared to the average score from tumor tissue with one (p = 0.01) or two copies (p = 0.03) of the PTEN gene. STAT1 expression and PTEN gene copy numberPTEN copy numberMean STAT1 in StromaMean STAT1 in Tumor>2 copies0.410.822 copies0.510.651 copy0.600.700 copies0.850.85

These results suggest that the STAT1 pathway may be influenced by PTEN loss in this tumor, suggesting that the JAK/STAT inflammatory pathway may have future clinical applications in PCa.

Citation Format: Thiago Vidotto, Clarissa G. Picanço-Albuquerque, Fabiano P. Saggioro, Rodolfo Borges dos Reis, David Robert Siemens, Madhuri Koti, Jeremy A. Squire. STAT1 expression in the tumor-stroma microenvironment is influenced by loss of PTEN in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 716.