bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4775: PTHrP regulate CSC and promote EMT in the PyMT model of breast cancer progression
Authors: Zhang Rui , Li Jiarong , Karaplis Andrew C. , Kremer Richard
Published: July 2017

PTH-related peptide (PTHrP) was first discovered in cancer patients as the primary cause of humoral hypercalcemia of malignancy (HHM). We previously showed that PTHrP ablation in the MMTV-PyMT murine model of breast cancer progression can dramatically prolongs tumor latency, slows tumor growth & metastases suggesting that PTHrP may regulate cancer stem cells (CSCs). CSCs & epithelial-mesenchymal transition (EMT) have been recognized as driving forces in tumor initiation & progression, & have been characterized previously using ALDH & CD49/EpCAM markers. Here, we aim to determine the role of PTHrP in CSCs & EMT regulation. For this purpose, we analyzed tumor cells derived from Pthrpflox/flox;Cre- (Control) & Pthrpflox/flox;Cre+ (KO) tumors. Cells were incubated with ALDH reagent followed by CD49f/EpCAM antibody labelling & subjected to FACS analysis. Using CD49f/EpCAM labelling, cells separated into two distinct populations, luminal progenitor cells (CD49f+EpCAM+) & EMT cells (CD49f+EpCAM-). In control tumors, ~85% of cells were characterized as luminal progenitor and ~15% as EMT. In contrast, in KO tumors, ~97% of cells were characterized as luminal progenitor whereas only ~2% were EMT. Using ALDH staining alone, ~8% & ~10% of the cells from the control & KO tumor were ALDH+, respectively. Furthermore, the majority of the ALDH+ cells were predominately contained within the luminal progenitor population in both the control & KO tumors cells. Next, we confirmed the dividing capacity of the two cell populations using a lipophilic fluorescent dye, PKH26. As expected, luminal progenitor cells exhibited a fast-dividing phenotype, whereas EMT cells displayed a slow dividing phenotype. However, using mammosphere assay, we did not see any significant difference in mammosphere forming efficiency between control & KO tumor. In summary, PTHrP ablation significantly diminishes the EMT population in KO compared to the control tumor. In conclusion, PTHrP may shift the tumor cell phenotype towards EMT, which suggests a mechanism underlying PTHrP - driven metastases.

Citation Format: Rui Zhang, Jiarong Li, Andrew C. Karaplis, Richard Kremer. PTHrP regulate CSC and promote EMT in the PyMT model of breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4775. doi:10.1158/1538-7445.AM2017-4775
Abstract 478: Regulation of cross talk between AR and wnt/beta-catenin pathways in prostate cancer
Authors: Plaga Alexis , Shukla Girish C.
Published: July 2017

Prostate Cancer (PCa) is the second most commonly diagnosed cancer in the United States with 180,890 males diagnosed and is predicted to cause approximately 26,000 deaths in 2016. The current treatment of androgen deprivation therapy (ADT) initially depletes circulating androgens; however, intratumoral androgens rescue androgen receptor (AR) signaling and promotes the development of castration resistant prostate cancer (CRPC). The dysregulation of the Wnt/β-catenin signaling pathway has been implicated in the development of many cancers including PCa. The disruption of this signaling leads to the stabilization of β-catenin which upregulates many genes involved in tumorigenesis. Additionally, β-catenin acts as AR cofactor. Our previous studies show that AR is a direct target of miR-644a. We hypothesized that targeting the expression of both the β-catenin pathway and AR by conventional drugs and/or tumor suppressor miR-644a would have synergistic therapeutic benefits. In this

study, we are investigating miR-644a mediated posttranscriptional downregulation of GSK3-β and β-catenin in the wnt/β-catenin signaling pathway. The effect of miR-644a in combination with inhibitors (β-catenin or GSK3-β) was also assessed using proliferation assays and a significant downregulation was observed. We will further study the posttranscriptional effects of tumor suppressor miR-644a on the wnt/β-catenin pathway and also the processing and regulation of miR-644a in PCa.

Citation Format: Alexis Plaga, Girish C. Shukla. Regulation of cross talk between AR and wnt/beta-catenin pathways in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 478. doi:10.1158/1538-7445.AM2017-478
Abstract 479: Modeling miRNA induced silencing in breast cancer with PARADIGM
Authors: Sedgewick Andrew J. , Benos Panayiotis V. , Rabizadeh Shahrooz , Soon-Shiong Patrick , Vaske Charles J.
Published: July 2017

Introduction: MicroRNAs play an important role in regulation of gene expression and are known biomarkers for breast cancer as well as other malignancies. PARADIGM is a pathway based algorithm that allows for integration of multiple genomic data types with a curated pathway database to make pathway activity predictions. We added a model of gene silencing due to miRNA to the PARADIGM algorithm in order to study miRNA expression in a pathway context.

Results: We curated a set of 7751 miRNA-mRNA interactions from the intersection of 3 target prediction algorithms. These interactions involved 66 miRNA and 2814 mRNA transcripts. We ran this model on global DNA copy number, RNAseq and miRNAseq data from 697 patients in the TCGA breast cancer cohort, and studied changes in the interactions between miRNAs and their targets between different tumor subtypes. The median activity of the RNA-induced silencing complex (RISC) predicted by our model is significantly higher in Basal tumors than other subtypes. In addition, RISC activity is significantly associated with overall survival of patients with Luminal A tumors. The miRNA-target pairs with the largest correlation changes between Basal and Luminal A subtypes were enriched for putative oncogenes and oncomirs. The mRNA targets are involved in a number of important signaling pathways including PI3K-AKT, JAK-STAT, and Ras. Many of these highly differential links involved the miR-16 family of miRNAs which are known tumor suppressors. miR-16 shows significantly lower activity in basal tumors than other subtypes.

Conclusions: By looking at changes in miRNA-target links between tumor subtypes, our algorithm was able to identify both miRNAs and target genes involved in pathways relevant to breast cancer. Our predictions of overall RNA-induced silencing activity show prognostic value in both determining subtype and predicting overall survival within subtypes.

Citation Format: Andrew J. Sedgewick, Panayiotis V. Benos, Shahrooz Rabizadeh, Patrick Soon-Shiong, Charles J. Vaske. Modeling miRNA induced silencing in breast cancer with PARADIGM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 479. doi:10.1158/1538-7445.AM2017-479
Abstract 4792: Prmt6 promotes lung tumor growth via modulation of Ilf2 expression
Authors: Avasarala Sreedevi , Wu Pei-Ying , Scoyk Michelle Van , Baskaran Abinaya , Su Yanlin , David Odile , Winn Robert A. , Bikkavilli Kamesh
Published: July 2017

Lung cancer kills more people in the US than any other malignancy. High mortalities are in part due to the diagnosis at an advanced inoperable stage where the 5-year survival is only 4%. Therefore improving survival will require increased knowledge of genes that drive the complex mechanisms of lung cancer initiation and progression. Emerging data implicate novel roles for protein arginine methylation in lung cancers. In protein arginine methylation, nitrogen/s of arginine can be post-translationally modified via the addition of a methyl group, catalyzed by a class of enzymes, protein arginine methyl transferases (PRMTs). Protein arginine methyl transferase 6 (PRMT6) is a methyl transferase that modifies histone tails, and its expression is upregulated in lung cancers, correlating with a worse overall patient survival. However, the in vivo role of PRMT6 has never been examined. In this study, we show that targeted over-expression of PRMT6 in the mouse lungs (PRMT6 gain-of-function mouse model) results in an increased chemical carcinogen-induced lung tumorigenesis. We also performed proteomics on PRMT6-associated proteins from the mouse lung and identified Interleukin-enhancer binding protein 2 (ILF2) a.k.a. nuclear factor 45 (NF45) as a novel PRMT6 interacting protein. By using a battery of cell-based assays, we show that PRMT6 plays an essential role in the regulation of the pro-proliferative functions of ILF2. Therefore, targeting PRMT6/ILF2 axis might open new possibilities for the therapeutic intervention of lung cancer.

Citation Format: Sreedevi Avasarala, Pei-Ying Wu, Michelle Van Scoyk, Abinaya Baskaran, Yanlin Su, Odile David, Robert A. Winn, Kamesh Bikkavilli. Prmt6 promotes lung tumor growth via modulation of Ilf2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4792. doi:10.1158/1538-7445.AM2017-4792
Abstract 4799: Fibroblast growth factor receptor (FGFR) promotes progression of cutaneous squamous cell carcinoma
Authors: Khandelwal Alok R. , Rong Xiaohua , Moore-Medlin Tara , Ma Xiaohua , Warner Amelia , DiGiovanni John , Nathan Cherie-Ann O.
Published: July 2017

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin. It is the second-most commonly diagnosed form of skin cancer (16%) striking 200,000 Americans annually. Increased understanding of the mechanisms involved in pathogeneses of cSCC could identify means to prevent, inhibit and reverse this process. In our previous studies, inhibition of Fibroblast growth factor receptor (FGFR) significantly decreased Ultraviolet B-induced (UVB) epidermal hyperplasia and hyperproliferation in SKH-1 mice suggesting an important role of FGFR signaling in skin cancer prevention. However, the role of FGFR signaling in the progression of cSCC is not yet elucidated. Analysis of the expression of FGFR2 in cSCC cells and normal epidermal keratinocytes revealed overexpression and increased FGFR2 activity in cSCC cells. Tumor cell-specific overexpression of FGFR2 was detected in human cSCCs and in UVB-induced mouse cSCCs, whereas the expression of FGFR2 was low in premalignant lesions and normal skin. Further, treatment with a selective FGFR inhibitor; AZD4547 significantly decreased proliferation, migration, and invasion of cSCC cells in culture. The decrease in FGFR activation was associated with an inhibition of mTORC1 and mTORC2 signaling markers. Our studies provide mechanistic evidence for the role of FGFR2 in early progression of cSCC and identify FGFR as a putative therapeutic target in the treatment of skin cancer.

Citation Format: Alok R. Khandelwal, Xiaohua Rong, Tara Moore-Medlin, Xiaohua Ma, Amelia Warner, John DiGiovanni, Cherie-Ann O. Nathan. Fibroblast growth factor receptor (FGFR) promotes progression of cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4799. doi:10.1158/1538-7445.AM2017-4799
Abstract 4801: Collection of lung cancer patient-derived xenograft (PDX) models to evaluate precision therapeutic strategies
Authors: Molina-Pinelo Sonia , Melendez Ricardo , Suarez Rocío , García Laura , Ojeda Laura , Moreno Nicolas , Guijarro María V , Paz-Ares Luis , Ferrer Irene
Published: July 2017

Background: Translational cancer research needs appropriated preclinical models to discover predictive biomarkers and evaluate precision therapeutic strategies. Conventional available preclinical models have proven not been optimal to this end, since cell lines based xenografts do not reconstitute the complexity of human cancer and carry acquired mutations not found in the original tumor. For these reasons our aim was generate and completely characterize a collection of non-small cell lung cancer (NSCLC) PDX models useful for translational lung cancer research.

Materials and methods: Resected primary tumors from NSCLC patients were subcutaneous xenografted in athymic nude mice and expanded in successive groups of mice to get a perpetual live bank of each tumor. Every tumor, which successfully grew in mice, was analyzed by histologic and molecular techniques (NGS gene panel, exome and RNA Seq). Furthermore, a bank of frozen pieces of tumor was expanded and stored in order to generate later cohorts of tumor-bearing mice suitable for preclinical drug evaluation.

Results: Our collection consists on 42 PDX models (26 SCC, 12ADC, 3LCC). We have characterized all of them histologically and molecularly with a 22 genes NGS panel. Furthermore, we have analyzed the exome and transcriptome of 33 models (20 SCC, 10 ADC and 3 LCC). All PDX models mostly retain the principal histologic and molecular characteristics of their donors and recapitulate the heterogeneity of human lung tumors. In our PDX collection we have sufficiently represented all the NSCLC histology and the most relevant molecular alterations in lung cancer in order to perform precision medicine evaluation studies.

Conclusions: We have generated a collection of 42 PDX models of NSCLC characterized at the histological, genomic and transcriptomic level, which represents the most frequent histological and molecular subtypes of this type of lung cancer. This collection will be really useful to integrate drug screening with biomarker discovery and to evaluate precision therapeutic strategies. Our future aim will be to use this collection for biomarkers identification and preclinical evaluation of new therapeutic strategies targeted to bad prognostic lung tumors with suboptimal therapeutic approaches.

Citation Format: Sonia Molina-Pinelo, Ricardo Melendez, Rocío Suarez, Laura García, Laura Ojeda, Nicolas Moreno, María V Guijarro, Luis Paz-Ares, Irene Ferrer. Collection of lung cancer patient-derived xenograft (PDX) models to evaluate precision therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4801. doi:10.1158/1538-7445.AM2017-4801
Abstract 4804: Sex-specific differences in Benzo(a)Pyrene [B(a)P]-induced colon carcinogenesis
Authors: Harris Kenneth J. , Harris Kelly L. , Washington Mary K. , Amos-Landgraf James , Ramesh Aramandla
Published: July 2017

Colorectal cancer (CRC) is the third most common diagnosed cancer and the third leading cause of cancer-related deaths in the United States. It has also been reported that colon cancer incidence and mortality rates are higher in men than woman, but there is yet a determined mechanistic link to show the factors that underlie the sex-specific differences in CRC initiation and progression. Benzo(a)pyrene [B(a)P], a member of the polycyclic aromatic hydrocarbon (PAH) family of compounds is a well-characterized environmental toxicant that has been proven to be a major contributor to the development of sporadic colon cancer. Published studies indicate that Aryl Hydrocarbon Receptor (AhR), a receptor for [B(a)P], bind to estrogen receptor (ER) and negatively affect AhR-target gene transcription. This study aims to elucidate the sex-specific differences in B(a)P-induced colon cancer in adult Polyposis In the Rat Colon (PIRC) model. We hypothesize that sex-specific differences in B(a)P biotransformation modulates the formation of colon tumors in PIRC rats. Groups of female and male PIRC rats (n = 8) received sub-chronic exposure to 25, 50 and 100 µg B(a)P/kg body wt. via oral gavage for 60 days. Female and male rats that received no [B(a)P] treatment served as controls. [B(a)P] was shown to have no significant effect on body weight of these rats and female PIRC rats that received 25, 50 and 100 µg B(a)P/kg body wt. showed significant decrease in total polyp count when compared to males with respective treatments. Polyp sizes of female PIRC rats receiving 25, 50 and 100 µg B(a)P/kg body wt. were increased when compared to males respectively. Histopathological analysis of colon polyps revealed that female animals exhibited low-grade to no dysplasia while high-grade dysplasia was recorded in male animals treated with corresponding doses. Phase 1 enzyme, Cytochrome P450 isoform 1A1 (CYP1A1), and phase 2 enzyme, Sulfotransferase Family 1A Member 1 (SULT1A1), were downregulated in colon tissue of female PIRC rats receiving 25, 50 and 100 µg B(a)P/kg body wt. when compared to male counterparts. In future studies, by measuring the expression of other phase 1 and phase 2 drug metabolizing enzymes (DME), along with measuring circulating estrogen levels, analyzing [B(a)P] metabolite profile, and probing B(a)P-DNA interactions, we will provide insight into if and how estrogen receptor protects females from developing colon cancer. This research was funded by NIH grants 5RO1CA142845-04, 5R25GM059994-3, and G12MD007586-29.

Citation Format: Kenneth J. Harris, Kelly L. Harris, Mary K. Washington, James Amos-Landgraf, Aramandla Ramesh. Sex-specific differences in Benzo(a)Pyrene [B(a)P]-induced colon carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4804. doi:10.1158/1538-7445.AM2017-4804
Abstract 4806: Strategies to overcome the heterogeneity of tumor cells in breast cancer therapy
Authors: Calaf Gloria M. , Gallardo Marcela , Roy Debasish , Ponce-Cusi Richard
Published: July 2017

Breast carcinogenesis is a multistage process that involves mutations and alterations attributed to exposure to exogenous environmental substances and endogenous agents as female hormones. To overcome the heterogeneity of tumor cells in breast cancer therapy several strategies must be considered from the bench to clinical settings. It is proposed four strategies: Analysis to determine 1) Apoptosis, 2) Epithelial-mesenchymal transition (EMT), 3) CD44/CD24 gene and protein expression in cells derived from mammospheres and 4) MicroRNAs as miR34a and others by the effects of chemotherapeutic drugs (as pamidronate (Pam), 5-Fluorouracil (5-FU) and antioxidants as Curcumin (Cur) (diferuloylmethane) derived from Curcuma longa. Pam, a bisphosphonate is used in the treatment of breast cancer. 5-FU is a chemotherapeutic agent for the treatment of a variety of solid cancers that arrest cell cycle and induce apoptosis in cancer cells. We evaluated genes and proteins targeted by these drugs and antioxidants in a triple positive cell line, as MCF7 and a negative, MDA-MB-231 for hormonal receptors, respectively and in an in vitro breast cancer model induced by radiation and estrogen that was developed with a normal immortalized breast epithelial cell line, MCF-10F exposed to low doses of high LET (linear energy transfer) alpha particles (150 keV/μm) of radiation, and cultured in presence of 17β-estradiol. We used: i) MCF-10F, ii) Alpha3, a malignant non-tumorigenic, iii) Alpha5, a tumorigenic one and iv) Tumor2 cell line derived from Alpha5 injected into the nude mice. Previous results showed increased cell proliferation, anchorage independency, invasive capabilities and tumor formation in nude mice, microsatellite instability and loss of heterozygosity in chromosomes 6, 8, 11 and 17 and mutations of c-Ha-ras and Rho-A in Alpha5 and Tumor2 compared to the control MCF-10F and their counterparts. Pam, 5-FU and Cur inhibited migration and invasion in both cell lines, and also decreased c-Ha-ras, Rho-A, p53, and Cav-1 gene expression by RT-qPCR. These compounds also induced 1) apoptotic effect on Bcl-xL and Bax gene and protein expression and by flow cytometry; 2) changes in EMT markers such as Snail, Slug, Axl, 3) CD44/CD24 alterations and 4) target MicroRNAs as miR34a on different process. It can be concluded that strategies to overcome the heterogeneity of tumor cells can be used to interfere with genes involved in critical steps in breast carcinogenesis. Grant: Tarapacá University, Arica, Chile (GMC).

Short tittle: Strategies in breast cancer therapy

Citation Format: Gloria M. Calaf, Marcela Gallardo, Debasish Roy, Richard Ponce-Cusi. Strategies to overcome the heterogeneity of tumor cells in breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4806. doi:10.1158/1538-7445.AM2017-4806
Abstract 4820: Developing a xenograft human tumor model in immunocompetent mice
Authors: Basel Matthew T. , Narayanan Sanjeev , Ganta Chanran , Shrestha Tej B. , Pyle Marla , Bossmann Stefan H. , Troyer Deryl L.
Published: July 2017

Animal models are essential to preclinical cancer research and are used clinically to determine optimal treatment regimens, but current xenograft models are limited in their utility, especially due to the lack of a competent immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and, after birth, mice were challenged again with A375 cells to determine their ability to develop tumors. Intravenous injections of A375 cells after tolerization resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. The development of lung neoplasms was dependent on fetal tolerization; non-tolerized mice did not develop tumors after injection. Interestingly, subcutaneous injected cells did not form tumors, but this was shown to be due to an innate, non-adaptive immune response and did not create lasting rejection of tumor cells. This procedure was repeated with several different tumor lines to show the universal nature of the method: BxPC3 (human pancreatic cancer), M21 (human melanoma), HeLa (human cervical cancer), and MDA231 (human breast carcinoma).

Citation Format: Matthew T. Basel, Sanjeev Narayanan, Chanran Ganta, Tej B. Shrestha, Marla Pyle, Stefan H. Bossmann, Deryl L. Troyer. Developing a xenograft human tumor model in immunocompetent mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4820. doi:10.1158/1538-7445.AM2017-4820
Abstract 4833: Perfusion-based bioreactor culture of primary cancer tissue maintains tumor microenvironment complexity and allow in-vitro testing of immune blockade therapy
Authors: Muraro Manuele Giuseppe , Muenst Simone , Manfredonia Celeste , Mele Valentina , Daester Silvio , Tzankov Alexandar , Terracciano Luigi , Weber Walter , Spagnoli Giulio C. , Iezzi Giandomenica , Martin Ivan , Soysal Savas D.
Published: July 2017

In vitro culture of primary cancer tissue is still very limited and the generation of patient derived xenograft determine the loss of human-cancer associated stroma. In this context, the use of 3D in vitro systems based on human tissue may be an innovative system to be exploited for keeping the tumor microenvironment (TME) complexity of the tissue in vitro.

Freshly excised colorectal (CRC) and breast cancer (BrCa) specimens were fragmented and cultured in 3D “sandwich-like format” between porous collagen scaffolds under perfusion flow (U-CUP, Cellec Biotek AG). The maintenance of tumor and immune-infiltrating cells, survival and phenotypic characterization were histologically assessed. In a second step cancer treatment were tested.

U-CUP culture allowed the preservation, viability and expansion of tumor tissue with concomitant stromal and immune cells. Expanding cancer cells were viable after 10 and 21 days (CRC and BrCa, respectively). Administration of anti-ER treatment to Lumina A ER+ BrCa was associated with decreased expansion of cancer tissue into the scaffold after 21 days. The maintenance of immune-infiltrating cells allowed testing of immune blockade therapy. Administration of anti-PDL1 antibody, alone or in combination with anti-CTLA4, to the culture medium was associated with increased expression of markers of immune-activation (i.e. IFNγ) and decreased expression of immunosuppressive cytokine IL10.

Preserving malignant, interstitial and immunocompetent cells comprised in surgically excised tumor specimens might allow a direct evaluation of the effects of various treatments on the complex TME. This engineered in vitro model could allow animal-free testing and it could be extended as a platform allowing the testing of innovative approaches for the treatment of human malignancies. Our findings shed the light on a promising system for selecting personalized treatment based on a patient’s tumor specific microenvironment.

Note: This abstract was not presented at the meeting.

Citation Format: Manuele Giuseppe Muraro, Simone Muenst, Celeste Manfredonia, Valentina Mele, Silvio Daester, Alexandar Tzankov, Luigi Terracciano, Walter Weber, Giulio C. Spagnoli, Giandomenica Iezzi, Ivan Martin, Savas D. Soysal. Perfusion-based bioreactor culture of primary cancer tissue maintains tumor microenvironment complexity and allow in-vitro testing of immune blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2017-4833
Abstract 4838: Optimization of prostate cancer organoid culture methods to grow PDX and human biopsy tissue
Authors: Beshiri Mike L. , Tran Crystal , Sowalsky Adam G. , Nguyen Holly M. , Agarwal Supreet , Tice Caitlin M. , Corey Eva , Kelly Kathy
Published: July 2017

A major obstacle in the field of prostate cancer research is the limited capacity to culture prostate cancer-derived cells in vitro. There are relatively few established prostate cancer cell lines in use today. Those that are available are amenable to in vitro drug assays and mechanistic studies, but do not well-represent the genetic diversity of the disease, nor do they accurately predict in vivo response. Recently, organoid culture techniques have been developed for prostate cancer by the laboratories of Hans Clevers and Charles Sawyers that have increased our ability to grow metastatic tumor-derived prostate cancer cells in vitro. This represents a great step forward, but we are still restricted by the limited availability of biopsy tissue and the relatively low success rate (~20%) in establishing new organoid lines. The LuCaP series of patient-derived xenografts (PDXs) represents a well-characterized set of prostate cancer specimens that is genetically diverse, reflective of a range of treatment histories, and available to the research community. We have modified the organoid culture technique based upon systematic comparisons and analysis for organoid growth of the LuCaP PDXs. Our modified conditions represent an improvement upon the original growth conditions. We have successfully cultured 21 out of 24 LuCaPs attempted, for at least one generation, and 16 out of 24 are able to grow long-term over several generations. This includes 3 of the newest LuCaPs (167, 170.2, and 189.3) that we have genomically characterized. Comparison of the PDX and various organoid generations showed significant conservation of genomic changes ranging from 75-90% for nonsynonymous point mutations. The modified organoid culture method immediately and significantly increases the number of prostate cancer lines that are available by making the LuCaP PDXs accessible to in vitro culture. In addition to the PDXs, our method has improved the success for culturing metastatic samples grown from biopsies. We show here that 2 out of 3 patient-derived organoid lines that we have established, grow well in the modified conditions but poorly in the original culture conditions, the third grows equally well in both conditions.

Citation Format: Mike L. Beshiri, Crystal Tran, Adam G. Sowalsky, Holly M. Nguyen, Supreet Agarwal, Caitlin M. Tice, Eva Corey, Kathy Kelly. Optimization of prostate cancer organoid culture methods to grow PDX and human biopsy tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4838. doi:10.1158/1538-7445.AM2017-4838
Abstract 4839: Ephrin-A ligands regulate cutaneous tumor etiology and metastasis through cell autonomous and non-autonomous mechanisms
Authors: Zheng Ji , White Bethany Perez , Kaplan Nihal , Zhao Zhe , Blumenberg Miroslav , Getsios Spiro , Wang Bingcheng
Published: July 2017

Glycophosphatidylinositol-anchored ephrin-A ligands target EphA receptor tyrosine kinases (RTKs) to promote keratinocyte differentiation. Accordingly, genetic ablation of the major epidermal EphA subtype, EphA2, increases susceptibility to DMBA/TPA-induced cutaneous chemical carcinogenesis. Defining the corresponding role of ligands for EphA2 in skin cancer has been more cumbersome as the three ephrin-A genes (Efna1, Efna3, Efna4) are all prominently expressed in skin. We met this challenge by utilizing a triple Efna1/3/4 knockout (TKO) mice that reflects the pattern of reduced Efna gene expression found in mouse and human cutaneous SCCs. Skin tumors developed earlier and grew faster in mice lacking EphA2 or these three ephrin-A ligands. Interestingly TKO mice displayed accelerated malignant progression toward invasive SCC that metastasized to the lymph nodes and lungs by 25 weeks following DMBA-TPA treatment. Using keratinocyte culture models, we found that ephrin-A ligands act within the epidermis to limit keratinocyte migration in a manner that depends on their targeting of EphA2. We also illustrated a key role for ephrin-A ligands in the surrounding tumor microenvironment by re-introducing isogenic SCC cell lines into the skin of wild-type or TKO mice on congenic FVB background; tumor growth and metastasis was facilitated in mice lacking ephrin-A ligands. Importantly, the invasive phenotype of TKO mouse tumor cell lines was normalized by genetic reintroduction of either Efna1 or Efna3. Integrating our findings from human tumors, mouse models, primary cell cultures, and allograft models provides strong support for the notion that ephrins operate within tumor cells and also in the microenvironment to suppress skin tumor initiation and metastasis.

Note: This abstract was not presented at the meeting.

Citation Format: Ji Zheng, Bethany Perez White, Nihal Kaplan, Zhe Zhao, Miroslav Blumenberg, Spiro Getsios, Bingcheng Wang. Ephrin-A ligands regulate cutaneous tumor etiology and metastasis through cell autonomous and non-autonomous mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4839. doi:10.1158/1538-7445.AM2017-4839
Abstract 4843: ANKRD52 inhibited tumor metastasis through dephosphorylation of PAK1 in lung adenocarcinoma
Authors: Lin Yen-Fan , Liu Ying-Pu , Lee Ting-Fang , Wu Cheng-Wen
Published: July 2017

As a leading cause of cancer-related deaths, metastasis is the ultimate challenge in our effort to fight cancer as a life-threatening disease. A member of ankyrin repeat proteins, ankyrin repeat domain 52 (ANKRD52), is studied here to be involved in the regulation of lung adenocarcinoma metastasis. Ankyrin repeat proteins are known to function as a platform for protein-protein interactions, and ANKRD52 was shown to interact with protein phosphatase 6 (PP6). This study aimed to investigate the anti-metastatic roles of ANKRD52 in lung adenocarcinoma. Immunohistochemistry staining in lung cancer tissue revealed prolonged disease-free survival in high ANKRD52 expressing cases. Using lung cancer cell lines, ANKRD52 knockdowns showed significantly increased cell mobility while forced expressed ANKRD52 decreased cell mobility. Using mass spectrometry analysis, p21-activated kinases (PAK1) was identified to interact with ANKRD52. Upregulated PAK1 phosphorylation was detected in ANKRD52 or PP6 knockdowns. Our model demonstrated a novel anti-metastatic protein, ANKRD52, that regulates cell mobility through interactions with PP6 and dephosphorylation of PAK1.

Citation Format: Yen-Fan Lin, Ying-Pu Liu, Ting-Fang Lee, Cheng-Wen Wu. ANKRD52 inhibited tumor metastasis through dephosphorylation of PAK1 in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4843. doi:10.1158/1538-7445.AM2017-4843
Abstract 4844: CRISPR-generated BAP1 knockdown in uveal melanoma cell lines affects organotropism
Authors: Mahal Katharina , Brooks Kelly , Mandal Amit , Smith Matthew , Dhomen Nathalie , Marais Richard
Published: July 2017

Uveal melanoma (UM), a genetically distinct subtype of melanoma, arises from melanocytes located in the uveal tract of the eye, and is the most common intraocular cancer in adults. Clinically evident metastasis occurs in about 50% of patients, often several years after successful treatment of the primary tumour, with secondary disease occurring predominantly in the liver. In most cases surgical resection is not possible, and there are currently no effective chemotherapeutic treatments available once the metastatic disease has developed. Loss of the tumor suppressor and chromatin modifier BAP1 (BRCA1-associated protein 1) is linked with poor prognosis and high risk for developing liver metastasis. However, our knowledge about the processes involved in UM metastasis and the mechanisms that enable metastatic growth preferentially in the liver is limited.

We recently described a panel of isogenic BAP1 knockout/knockdown UM cell lines generated by CRISPR technology, which allowed us to study the cellular effects of BAP1 loss in comparison to parental UM cell lines. In mouse xenograft studies we were able to show that BAP1-loss of function (LOF) promoted metastatic growth preferentially in the liver, while the parental cells metastasized mainly to lymph nodes and lung.

A possible explanation for the observed liver tropism could lie in the alteration of protein secretion or receptor expression patterns upon BAP1 knockout that may result in an increased sensitivity to liver specific growth factors or cytokines. We are currently investigating gene expression and protein alterations resulting from BAP1 knockdown, and the reciprocal signaling between the CRISPR cell line pairs and liver cells such as hepatocytes and hepatic stellate cells in co-culture experiments. These studies will reveal a potential mechanism by which loss of BAP1 function mediates liver tropism in vivo and potentially provide new therapeutic avenues for patients at high risk of metastatic UM disease.

Citation Format: Katharina Mahal, Kelly Brooks, Amit Mandal, Matthew Smith, Nathalie Dhomen, Richard Marais. CRISPR-generated BAP1 knockdown in uveal melanoma cell lines affects organotropism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4844. doi:10.1158/1538-7445.AM2017-4844
Abstract 4845: Induction of aldolase C expression by NME1 may mediate metastasis suppressor activity in malignant melanoma
Authors: Pamidimukkala Nidhi , Leonard Katie , McCorkle Joseph , Zhang Qingbei , Mahurkar Anup , Shetty Amol , Kaetzel David
Published: July 2017

Although the metastasis suppressor activity of NME1 has been demonstrated in a number of settings of

human cancer, the underlying molecular mechanisms are not well understood. NME1 has two known

enzymatic activities, nucleoside diphosphate kinase (NDPK) and a 3’ to 5’ exonuclease. To determine

the relative contribution either of these activities has on the metastasis suppressor function of NME1,

our lab previously generated site directed mutants to abrogate one or both of the enzymatic activities

(Ma et al., 2004). These studies showed that disrupting the 3’ to 5’ exonuclease activity with the point

substitutions glutamate5-to-alanine (E5A) and lysine12-to-glutamine (K12Q) also significantly compromised

the metastasis suppressor function of NME1 (Zhang et al., 2011). To identify genes that are regulated by

NME1 and potentially modulate its metastasis suppressor activity, we compared the gene expression

profile induced by forced expression of wild-type NME to those obtained with the E5A and K12Q mutants.

Using the human Exon 1.0 Affymetrix microarray , we identified 13 genes whose expression was

modulated by wild-type NME1 expression, but not by the suppressor-deficient mutants, which were

designated as metastasis suppressor signature (MSS) genes. We are currently refining the list to genes

that have the highest clinical relevance through observing expression profiles of the MSS genes in

clinical datasets. Preliminary results of unsupervised hierarchal clustering of GEO melanoma dataset

GSE8401 showed that expression of the MSS gene, aldolase C (ALDOC), was significantly lower in a

subset of metastatic melanoma lesions compared to primary melanomas. Western blot and qPCR

analysis confirm that NME1 induces expression of ALDOC in both WM793 and M14 melanoma cell lines,

and that induction of ALDOC is dose-responsive to NME1 expression as observed in the WM1158

metastatic melanoma cell line. Further, NME1 induction of ALDOC is specific compared to the ALDOA

and ALDOB isoforms. Recent studies have indicated aldolase isoforms can modulate motile and invasive

phenotypes of cancer cells (Li et al., 2015 & Hu et al., 2016), suggesting regulation of ALDOC expression

may represent a key mechanism underlying the metastasis suppressor function of NME1. Current work

is focused on measuring the impact of NME1-induced expression of ALDOC on the metastatic potential

of melanoma cells in culture and in vivo.

Citation Format: Nidhi Pamidimukkala, Katie Leonard, Joseph McCorkle, Qingbei Zhang, Anup Mahurkar, Amol Shetty, David Kaetzel. Induction of aldolase C expression by NME1 may mediate metastasis suppressor activity in malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4845. doi:10.1158/1538-7445.AM2017-4845
Abstract 4846: The metastasis suppressor NME1 is recruited to DNA double strand breaks
Authors: Puts Gemma S. , Jarrett Stuart G. , Snyder Devin , Vincent Richard , Wang Ying , Leonard Katie , Portney Ben , Rassool Feyruz , Zalzman Michal , Kaetzel David M.
Published: July 2017

NME1 is a potent suppressor of metastasis, and reduced expression has been associated with aggressive melanoma. The nucleoside diphosphate kinase and 3’-5’ exonuclease (3’-5’ EXO) activities of NME1 protein both contribute to its metastasis suppressor activity. 3’-5’ exonucleases are important for proofreading during DNA repair and synthesis, suggesting a potential role for NME1 in maintenance of genomic stability in melanoma. Although impairment of DNA repair activity has been implicated in melanoma risk, its contribution to melanoma initiation and progression is not well understood. We have shown previously that both enzymatic activities of NME1 are critical for repair of UV-induced DNA damage via nucleotide excision repair. In the current study, chromatin immunoprecipitation was used to demonstrate recruitment of NME1 to DNA double-strand breaks (DSBs), representing the first evidence of direct recruitment of NME1 to sites of DNA damage. Induction of DSBs with gamma irradiation (γ-IR) or bleomycin promoted rapid physical association of NME1 with γH2AX, ATM, NBS1 and RAD50. In addition, treatment of melanoma cells with γ-IR triggered a physical association of NME1 with XRCC4, an effector of the non-homologous end-joining (NHEJ) pathway. We are currently investigating whether knockout of NME1 induces a switch from NHEJ to the homologous recombination (HR) pathway, a slower but higher fidelity form of DSB repair, using plasmid repair assays. We are also using plasmid rescue to study the role of NME1 on the fidelity of NHEJ repair. Overall, our study indicates for the first time direct participation of NME1 in a molecular mechanism of DSB repair in both normal and transformed cells. In addition, these data suggest a novel mechanism of metastasis suppressor function for NME1, with loss of NME expression in tumor cells conferring genetic instability and enhanced metastatic potential.

Citation Format: Gemma S. Puts, Stuart G. Jarrett, Devin Snyder, Richard Vincent, Ying Wang, Katie Leonard, Ben Portney, Feyruz Rassool, Michal Zalzman, David M. Kaetzel. The metastasis suppressor NME1 is recruited to DNA double strand breaks [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4846. doi:10.1158/1538-7445.AM2017-4846
Abstract 4849: ELMO1 promotes metastasis in colorectal cancer cells via activation of MAPK/ERK signaling pathway
Authors: Zheng Xiao-bin , Zhou Chi , Cheng Hai-chun , Hu Tuo , Liu Hua-shan , Liu Xuan-hui , Wu Xian-rui , Wang Feng-wei , Chen Yu-feng , Wang Jian-ping , He Xiao-sheng , Lan Ping
Published: July 2017

Background and Aim: Engulfment and cell motility 1 (ELMO1) has been linked to the invasive phenotype of various types of cancer cells such as glioma cells, rhabdomyosarcoma cells, ovarian cancer cells and so on. However, the biological functions of ELMO1 in colorectal cancer (CRC) cells remains unclear. The aim of this study is to evaluate the role of ELMO1 in the metastasis in CRC.

Methods: The expression level of ELMO1 was detected using quantitative RT-PCR and western blot in 9 CRC cell lines and 1 human colon epithelial cell line NCM460. ELMO1 was overexpressed via lentiviral vector system and silenced using specific small interference RNA (si-ELMO1) in HCT116 and SW620. Then wound-healing, migration assay and invasion assay were carried out to investigate the function of ELMO1 in CRC cells. Western blot was performed to detect the change of signaling molecule extracellular signal-regulated kinase 1/2 (ERK1/2) following the down-regulated or up-regulated expression of ELMO1.

Results: ELMO1 expression was inhibited by si-ELMO1 and was overexpressed by ELMO1-cDNA in HCT116 and SW620. Ectopic expression of ELMO1 in HCT116 and SW620 was shown to promote migration and invasion, while inhibited ELMO1 expression suppressed the capacity of cell migration and invasion. Furthermore, ELMO1 induced migration and invasion in CRC cells was found to be associated with the activation of MAPK/ERK pathway.

Conclusions: High expression of ELMO1 was found to be associated with the metastasis via the activation of MAPK/ERK signaling pathway. This finding provides a molecular basis for the role of ELMO1 in the progression of CRC, which may suggest a novel target for the treatment of CRC.

Citation Format: Xiao-bin Zheng, Chi Zhou, Hai-chun Cheng, Tuo Hu, Hua-shan Liu, Xuan-hui Liu, Xian-rui Wu, Feng-wei Wang, Yu-feng Chen, Jian-ping Wang, Xiao-sheng He, Ping Lan. ELMO1 promotes metastasis in colorectal cancer cells via activation of MAPK/ERK signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4849. doi:10.1158/1538-7445.AM2017-4849
Abstract 485: Epigentic silencing of miR-142-3p promotes metastasis by targeting ZEB2 in NPC
Authors: Li Yingqin , Liu Na , Ma Jun
Published: July 2017

Metastasis is the major cause of cancer-related death in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms governing metastasis remain poorly understood in NPC. Here, we demonstrated that miR-142-3p was downregulated and significantly suppressed NPC cell migration, invasion in vitro and in vivo. We identified zinc finger E-box binding homeobox 2 (ZEB2) as a direct target of miR-142-3p, and its overexpression rescued the inhibitory effects of miR-142-3p in NPC. Importantly, miR-142-3p downregulation was associated with DNA methylation within the miR-142 locus. Enhancer of zeste homologue 2 (EZH2) suppressed miR-142 methylation and miR-142-3p expression. Systemic reversal of miR-142 silencing suppressed lung metastasis of NPC cells. Furthermore, miR-142 hypermethylation was associated with poor metastasis-free survival. This study demonstrates a suppressive role of miR-142-3p in metastasis and uncovers an epigenetic mechanism for miR-142-3p silencing in NPC, suggesting a potential prognostic factor and therapeutic target to combat metastasis in NPC.

Note: This abstract was not presented at the meeting.

Citation Format: Yingqin Li, Na Liu, Jun Ma. Epigentic silencing of miR-142-3p promotes metastasis by targeting ZEB2 in NPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 485. doi:10.1158/1538-7445.AM2017-485
Abstract 4862: SORBS1-AHNAK complex regulates metastatic properties of cancer
Authors: Cho Woo-Cheol , Jang Ji-Eun , Ku Ja-Lok
Published: July 2017

Purpose: CAP encoded by SORBS1, Vinexin and ArgBP2 is participated in the SoHo family of proteins. The SoHo protein interacts with different signaling molecules and involves with cell migration, and has been implicated in a variety of cellular processes including insulin-stimulated glucose transport. CAP has specific role in the stabilization of adhesion complexes and the consequent effect of cell migration. It is possible that CAP or complex between CAP and other proteins induces change of cancer cell characters. We assumed that SORBS1 has substantial role of cell proliferation, migration and interaction with other subcellular proteins.

Methods and materials: To elucidate endogenous role of SORBS1, colorectal cancer cell lines were manipulated by pcDNA, siRNA and shRNA in some colorectal cancer cell lines. Function of SORBS1 as regulator of cancer proliferation was demonstrated by cell cycle assay. And several methods including western blotting, cell migration assay and immunoprecipitation assay were performed for determining role of SORBS1 related other metastatic properties.

Conclusion: Colony formation ability and proliferation were induced by SORBS1-overexpression in HT29 cell line. In contrast, transient suppression of SORBS1 inhibits cell proliferation. Moreover, when SORBS1 was constantly suppressed in HCT-116 and SNU-C4 cell line, cell migration was impeded. According to immunoprecipitation data, we chose the AHNAK protein as the convincing candidate protein related with SORBS1. An interaction of SORBS1 and AHNAK was observed in both cytoplasm and nucleus. Expression level of SORBS1 and AHNAK was also negative correlation in extraction of cytoplasm and nucleus.

SORBS1 and AHNAK bind each other as form of protein complex, however, AHNAK known as nucleoprotein is abundant in both cytoplasm and nucleus when SORBS1 is suppressed. Consequently, these results indicate that SORBS1 has a key role of cancer cell growth and invasiveness through regulation of AHNAK expression and degradation.

Note: This abstract was not presented at the meeting.

Citation Format: Woo-Cheol Cho, Ji-Eun Jang, Ja-Lok Ku. SORBS1-AHNAK complex regulates metastatic properties of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4862. doi:10.1158/1538-7445.AM2017-4862