bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 570: Long-term efficacy and downstream mechanism of anti-Annexin A2 monoclonal antibody in a pre-clinical model of aggressive human breast cancer
Authors: Sharma Mahesh C. , Tuszynski George P. , Blackman Marc R. , Sharma Meena
Published: July 2016

Abstract:

There are considerable direct evidences to support that calcium binding protein ANX A2 is a potential target for aggressive breast cancer. The most compelling evidence is based on the fact that ANX A2 overexpress specifically in aggressive triple negative human breast cancer (TNBC) cell lines as well as in human breast cancer tissues. Previously, we and others have demonstrated a unique role of ANX A2 in cancer invasion including breast cancer. Moreover we demonstrated that anti-ANX A2 monoclonal antibody (anti-ANX A2mAb)-mediated immunoneutralization of ANX A2 inhibited invasive human breast growth in a xenograft model. We further evaluated long-term effect of multiple treatments of anti-ANX A2mAb and its mechanism of inhibition of human breast tumor growth. We now show that three treatments of anti-ANX A2mAb showed significant inhibition of breast tumor growth in immunodeficient mice and anti-tumor response was started from day 94. After treatments, we followed tumor growth for 172 days and our results demonstrated 67% inhibition of tumor growth without detectable adverse effects. Biochemical analysis demonstrated anti-ANX A2mAb treatment caused significant inhibition of tissue plasminogen activator (tPA) synthesis in the tumor microenvironment. This led to disruption of plasmin generation that consequently inhibited activation of MMP-9 and MMP-2. These results suggest that ANX A2 plays an important role in aggressive breast tumor growth by regulating proteolytic pathway in the tumor microenvironment. ANX A2 may represent a new target for the development of therapeutics for the treatment of aggressive breast cancer.

Citation Format: Mahesh C. Sharma, George P. Tuszynski, Marc R. Blackman, Meena Sharma. Long-term efficacy and downstream mechanism of anti-Annexin A2 monoclonal antibody in a pre-clinical model of aggressive human 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 570.
Abstract 593: Highly cytotoxic EGFR/CD16A TandAbs specifically recruit NK cells to potently kill various types of solid tumors
Authors: Ellwanger Kristina , Reusch Uwe , Fucek Ivica , Weichel Michael , Rajkovic Erich , Treder Martin
Published: July 2016

Constitutive EGFR activation through amplification or dysregulation plays an important role in the pathophysiology of numerous solid cancers, such as colorectal cancer (CRC), non-small cell lung cancer (NSCLC) or squamous cell carcinomas of the head and neck (SCCHN), thus providing a strong rationale for the development of therapeutic strategies targeting EGFR. Different therapeutic approaches have been approved for treatment of such cancers, including tyrosine kinase inhibitors that interfere with signal transduction by blocking the kinase domain or monoclonal antibodies (mAbs) that prevent EGFR ligand binding, dimerization and activation. However, despite demonstrated clinical efficacy, intrinsic or acquired resistance to such treatments has been described for a larger number of patients.

Natural killer cells (NK-cells) play a central role in the innate immune system and have the capacity to destroy virally-infected or neoplastic cells. To specifically utilize the cytotoxic potential of NK-cells for the elimination of EGFR+ cancer cells, we developed tetravalent bispecific EGFR/CD16A NK-cell TandAbs, with two binding sites for EGFR being expressed on tumor cells, and two binding sites for CD16A expressed on NK-cells.

Using antibody phage display technologies, we identified high affinity scFvs recognizing conformational epitopes in the extracellular domain of EGFR that were different to those targeted by other therapeutic antibodies. We engineered a set of bispecific EGFR/CD16A TandAbs and analysed their binding, thermostability and cytotoxic properties in a panel of in vitro assays. TandAbs containing our EGFR-specific domain 21 were highly potent in cytotoxicity assays towards endogenously EGFR-expressing tumor cell lines or transfected CHO cells with single digit picomolar or subpicomolar EC50 values. In contrast to comparator NK-cell recruiting TandAbs containing the Fv sequences from cetuximab, the TandAbs containing our EGFR-binding domain 21 did not exhibit any signs of temperature-induced instability or aggregation in thermostability studies.

Taken together, our data suggest that EGFR/CD16A TandAbs are novel, highly potent drug candidates suitable for the treatment of EGFR-overexpressing malignancies and overcoming resistance to other therapeutic agents.

Citation Format: Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Erich Rajkovic, Martin Treder. Highly cytotoxic EGFR/CD16A TandAbs specifically recruit NK cells to potently kill various types of solid tumors. [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 593.
Abstract 594: Antibodies targeting LAG-3, TIM-3, KIR cloned from healthy human donors
Authors: Estelles Angeles , Stephenson Robert , Liu Keyi , Lomongsod Evelene , Ngyen Da , Zhzng Jianzhong , Yang Yifan , Heideker Manfred , Kauvar Lawrence , Ryser Stefan , Gishizky Mikhail L.
Published: July 2016

The immunosurveillance/immunoediting theory postulates that tumor growth is repressed in healthy individuals through the activation of adaptive and innate immune mechanisms. The clinical success of antibodies that target inhibitory checkpoint pathways of the immune system (i.e. CTLA-4, PD-1) has provided compelling evidence in support of this theory and raises the question of whether antibodies targeting these, or other inhibitory immune cell modulators (ICM), are present in apparently healthy individuals. The present studies were designed to answer this question by probing the circulating memory B-cell compartment that represents a historic record of an individual's lifelong adaptive immune response.

Methods: Blood from donors, with no known cancer history, was screened for the presence of anti-ICM binding memory B-cells targeting LAG-3, TIM-3, B7-H3, KIR, and VISTA, using Trellis's established CellSpotTM technology that can detect the presence of antigen specific memory B-cells at a frequency of 0.1 per 100,000 memory B-cells.

Results: Circulating memory B-cells targeting ICM's were present in all apparently healthy individuals tested (20 total). There was substantial variation as to which ICM's were recognized within each individual across this sample population. Antibodies cloned from these rare B-cells were of IgG class and exhibited low to sub-nM affinity toward the specific ICM. These high affinity antibodies bound their respective antigens on activated T-cells and were able to augment immune stimulatory cytokine release in cellular assays. Data from ongoing studies will be presented.

Conclusion: These studies demonstrate that apparently healthy individuals produce high affinity, pharmacologically active anti-ICM antibodies. Thus, healthy individuals represent a previously unrecognized source of antibodies that have undergone natural selection and hold particular promise as potential therapeutic agents for the treatment of cancer.

Citation Format: Angeles Estelles, Robert Stephenson, Keyi Liu, Evelene Lomongsod, Da Ngyen, Jianzhong Zhzng, Yifan Yang, Manfred Heideker, Lawrence Kauvar, Stefan Ryser, Mikhail L. Gishizky. Antibodies targeting LAG-3, TIM-3, KIR cloned from healthy human donors. [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 594.
Abstract 596: Optimization of lead antibody selection for XMT-1522, a novel, highly potent HER2-targeted antibody-drug conjugate (ADC)
Authors: Bodyak Natasha , Yurkovetskiy Alex , Gumerov Dmitry R. , Xiao Dongmei , Thomas Joshua D. Thomas D. , Poling Laura L. , Qin LiuLiang , Yin Mao , DeVit Michael J. , Park Peter U. , Lee Winnie , Prinz Bianka , Bergstrom Donald A. , Lowinger Timothy B.
Published: July 2016

XMT-1522 is an anti-HER2 ADC that incorporates HT-19, a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery. Several parameters such as cell binding, internalization rate, cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) downstream signaling, affinity maturation, NHP cross-reactivity, normal human tissue cross-reactivity and in vivo efficacy were used to screen a wide range of antibodies to select a lead candidate optimized for use in ADC applications. In addition HT-19 was selected to be non-competitive for HER2 binding with existing therapies - trastuzumab or pertuzumab, to allow for potential combinability. In vivo efficacy as an ADC did not directly correlate with typical parameters used in antibody screening cascade such as in vitro cytotoxicity, internalization or binding affinity. The lead antibody underwent affinity maturation, and despite increases in affinity, affinity maturation significantly decreased the in vivo efficacy of the ADC in vivo xenograft models. This phenomenon was observed in all the antibody hits. HT-19 showed antibody-dependent cell-mediated cytotoxicity (ADCC) activity. When administered as a single agent in NCI-N87 gastric cancer xenograft model it had biological activity at 20 mg/kg as well as at 3 mg/kg. Consistent with the hypothesis that a non-competitive ADC is combinable with current anti-HER2 regimens, the combination of XMT-1522 with trastuzumab and/or pertuzumab showed more rapid internalization, more complete HER2 degradation, and significantly great anti-tumor activity in the NCI-N87 gastric cancer xenograft model relative to XMT-1522 alone or the combination of pertuzumab + trastuzumab.

Citation Format: Natasha Bodyak, Alex Yurkovetskiy, Dmitry R. Gumerov, Dongmei Xiao, Joshua D. Thomas D. Thomas, Laura L. Poling, LiuLiang Qin, Mao Yin, Michael J. DeVit, Peter U. Park, Winnie Lee, Bianka Prinz, Donald A. Bergstrom, Timothy B. Lowinger. Optimization of lead antibody selection for XMT-1522, a novel, highly potent HER2-targeted antibody-drug conjugate (ADC). [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 596.
Abstract 6: Metabolomics of Gleason score in prostate tumor tissue and serum
Authors: Penney Kathryn , Tyekucheva Svitlana , Loda Massimo
Published: July 2016

Background: Gleason score is currently the best clinical predictor of prostate cancer specific-mortality. Identifying biomarkers associated with Gleason score can improve prediction and advance the understanding of the biology of aggressive disease. Using gene expression data, we previously determined that metabolism pathways, including pyrimidine, propanoate, and beta-alanine, were differentially expressed in high and low grade tumors. The goal of this study is to identify metabolites that differ in low and high Gleason score tumors and in blood from these patients.

Methods: Metabolic profiling was performed on Gleason score 6 (n = 53) and Gleason score ≥8 (n = 32) radical prostatectomy tumor tissue specimens, and on serum samples from the time of diagnosis (n = 30 Gleason score 6 and n = 19 Gleason score ≥8) from the DF/HCC Prostate Cancer SPORE Cohort. Samples were prepared for analysis by Metabolon, Inc. with their standard solvent extraction method to recover small molecules. Metabolites were identified and quantified by gas and liquid chromatography and mass spectrometry. Missing values were calculated using k-nearest neighbor imputation, and sample values were normalized across batches. The metabolite levels were compared across Gleason score 6 and Gleason score ≥8 tumors and serum using a Wilcoxon test.

Results: In the tumor tissue, 207 metabolites were identified. Of these, 30 were present in significantly different amounts in high and low Gleason score tumors, including previously reported citrate (p = 0.002) and spermine (p = 0.003). In a pathway analysis, the propanoate pathway was significantly different in high and low Gleason (p = 0.03), but not the pyrimidine or beta-alanine pathways (p>0.05). In serum, 15 individual metabolites had significantly different levels, including uridine (p = 0.008) and N-acetylserine (p = 0.0001).

Conclusions: Metabolite levels do differ in high and low Gleason score prostate tumor tissue. The novel serum results suggest that metabolites in blood may serve as biomarkers of tumor differentiation. Our ongoing research will build prediction models, and will determine if the blood metabolites can indicate the presence of high-Gleason score tumor not detected at biopsy and could serve as biomarkers for monitoring disease progression in active surveillance patients.

Citation Format: Kathryn Penney, Svitlana Tyekucheva, Massimo Loda. Metabolomics of Gleason score in prostate tumor tissue and serum. [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 6.
Abstract 606: Similar 3D pharmacodynamic (3D-PD) responses of human tumor spheroids and xenografts to topoisomerase 1 inhibitor-induced DNA damage
Authors: Evans David M. , Delosh Rene , Laudeman Julie , Ogle Chad , Reinhart Russell , Selby Michael , Thomas Silvers , Kinders Robert , Navas Tony , Lawrence Scott , Monks Anne , Rapisarda Annamaria , Parchment Ralph E. , Doroshow James H. , Teicher Beverly
Published: July 2016

Three-dimensional (3D) cultures have been proposed as higher fidelity models of in vivo tumors than 2D cultures. To determine whether this idea extends to drug pharmacodynamics, we examined whether 3D cultures of the human melanoma line A375 (grown as tumor spheroids) could replicate the timing and magnitude of the nuclear γH2AX response to the topoisomerase 1 inhibitor, topotecan observed in A375 tumor xenografts (Kinders et al, Clin Can Res 2010). The appearance of γH2AX-positive nuclear foci has been used as a biomarker for drug- and radiation-induced double-strand breaks in DNA; we reported previously that treatment of nu/nu mice harboring A375 xenografts with a single-dose of topotecan induced nuclear γH2AX foci in a dose- and time-dependent manner, which peaked 4 hours after drug administration. A375 spheroids were generated by seeding cells into ULA U-bottom plates; 4 hours of exposure to 0.1 μM topotecan elicited a γH2AX signal in these 3D cultures while cell viability and intracellular ATP levels remained unchanged, indicating a DNA damage repair response at the maximally tolerated topotecan concentration for human hematopoietic cells (Erickson-Miller et al, Can Chemo Pharmacol 2009). Extending drug exposure to 24 hours caused substantial loss of viable cells (calcein AM+) and 50% decline in ATP levels but no further increase in γH2AX. In contrast, the HT29 tumor line was refractory to topotecan in vivo, and exposing 3D cultures of HT-29 spheroids to 0.1 μM topotecan for 24 hours elicited a strong nuclear γH2AX biomarker response in only a small fraction of cells at the surface of the spheroids. Longer exposure durations or supra-pharmacological concentrations (1 μM) of topotecan were required to achieve a strong nuclear γH2AX response in HT-29 spheroids. These results support the hypothesis that the 3D pharmacodynamics (PD) of drug response is similar to PD drug response in vivo for the camptothecin class of topoisomerase 1 inhibitors. Funded by NCI Contract No. HHSN261200800001E.

Citation Format: David M. Evans, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Michael Selby, Silvers Thomas, Robert Kinders, Tony Navas, Scott Lawrence, Anne Monks, Annamaria Rapisarda, Ralph E. Parchment, James H. Doroshow, Beverly Teicher. Similar 3D pharmacodynamic (3D-PD) responses of human tumor spheroids and xenografts to topoisomerase 1 inhibitor-induced DNA damage. [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 606.
Abstract 610: Effect of scaffold and growth factors on anti-cancer drug screening with multicellular spheroids: mimicking in vivo response
Authors: Masuda Norio , Rahman M. Mamunur , Arai Kazuya , Mizuno Atsushi , Itoh Manabu
Published: July 2016

Background:

For in vitro cancer research, cancer microenvironment is being increasingly recognized as a key factor. In the past, several 3D cell culture models have been tested as the powerful method for reproducing cancer microenvironment. However, in some cases, the growth and drug sensitivity of cells grown on anchorage-independent 3D culture models have been different to that of cells grown in vivo. Further, development of the 3D culture model including the physical (the cell-ECM interaction (scaffold)) and chemical factor (cytokine) are essential for mimicking cancer microenvironment in vitro. Therefore, we explored the effect of the growth factor dependent proliferation and drug sensitivity in scaffold/scaffold-free culture models, and try to understand which method is mimicking in vivo response due to stimulation and suitable for anti-cancer drug development.

Methods:

We cultured lung cancer, breast cancer, pancreatic cancer and ovarian cancer cell line on monolayer, Matrigel (ECM-embedded 3D culture, scaffold type), NanoCuluture Plate (NCP, a gel-free scaffold type 3D culture plate) and Low adhesion round-bottom plate (scaffold-free 3D culture plate). For growth curve experiment upon addition of growth factors (EGF, HB-EGF, FGF, HRG), we evaluated cell viabilities by ATP assay in several cancer cell lines. We examined drug sensitivities of cancer cell lines against clinically active anti-cancer agents by addition of growth factors.

Results:

Growth factor promoted the proliferation of cancer cell lines cultured on NCP while it was not enhanced the cell growth when cultured on 2D, Matrigel and Low adhesion round-bottom plate. In drug response, the cells grown on NCP by growth factor stimulation was more sensitive to drug than 2D and other 3D culture models.

Conclusion:

Differences of the cell growth and drug sensitivity were appeared by effect of scaffold and growth factor. Growth factor dependent cell proliferation and drug sensitivity on NCP were similar to in vivo behavior (1). These results demonstrated that scaffold and growth factor are the novel approach to be mimicking the cancer microenvironment in vitro. Therefore, it is obvious that NCP can be used as a suitable 3D culture model for mimicking in vivo response and anti-cancer drug development.

1. J Cell Sci. 2009 Dec 1;122(Pt 23):4277-86.

Citation Format: Norio Masuda, M. Mamunur Rahman, Kazuya Arai, Atsushi Mizuno, Manabu Itoh. Effect of scaffold and growth factors on anti-cancer drug screening with multicellular spheroids: mimicking in vivo response. [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 610.
Abstract 612: Identification of selective inhibitors of diffuse-type gastric cancer cells by screening of annotated compounds
Authors: Shimada Shu , Akiyama Yoshimitsu , Fukamachi Hiroshi , Yuasa Yasuhito , Tanaka Shinji
Published: July 2016

Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and a poor prognosis. We have reported an E-cadherin/p53 double conditional knockout (DCKO) mouse line as the first genetically engineered one, which morphologically and molecularly recapitulates human DGC. We have also established mouse gastric cancer (GC) cell lines from DGC of the DCKO mice, demonstrating strong resistance to cytotoxic anti-cancer agents and high tumorigenic property when subcutaneously injected to nude mice. Then, we performed a synthetic lethal screening of 1535 annotated chemical compounds, and identified 27 candidates with selective inhibitory activity against the GC cell lines. These candidate drugs were classified into some classes, and members of the most potent class specifically attenuated cell proliferation of the GC cell lines by induction of apoptosis as well as suppressing tumor growth in vivo. Expectedly, E-cadherin-mutant and -low human gastric cancer cell lines were more sensitive to them in contrast to E-cadherin-intact ones. These findings may lead to development of novel drugs and discovery of potential targets in human DGC.

Citation Format: Shu Shimada, Yoshimitsu Akiyama, Hiroshi Fukamachi, Yasuhito Yuasa, Shinji Tanaka. Identification of selective inhibitors of diffuse-type gastric cancer cells by screening of annotated 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 612.
Abstract 613: Astrocytes and endothelial colony forming cells (ECFCs) influence the migration and drug response of glioblastoma cells in a 3D culture model
Authors: Herrera-Perez Marisol , Fishel Melissa , Harbin Sherry , Rickus Jenna
Published: July 2016

Glioblastoma, a brain cancer with low prognosis, is characterized by rapid infiltration and drug resistance that leads to tumor reappearance. Cell-extrinsic factors, such as the development of a supportive microenvironment and synergistic relationships between normal cells and GBM play an important role in the tumor successful progression. Despite the importance of the microenvironment as a regulator of tumor response, there is a lack of predictive in vitro models that represent the dimensionality and characteristics of the glioma microenvironment. To this end, we developed a 3D in vitro model that recapitulates the composition and physical features of the GBM microenvironment by generating a composite matrix of a hyaluronan supported by collagen. Human astrocytes and endothelial colony forming cells (ECFCs) were incorporated into the matrix and co-cultured with GBM cells to evaluate the effect of normal cells on the GBM 3D cell migration and drug response. Time-lapse microscopy was used to evaluate the 3D migration distance, velocity and persistence of GBM cell lines GBM10, GBM43 and GBAM1 (CD133+) during 18h after drug treatment with temozolomide and a STAT3-inhibitor. Survival of GBM cells was also evaluated 18h and 72 h after drug treatment. All results were compared to standard monolayer culture. Presence of astrocytes and ECFCs increased GBM net migration distance and velocity in the 3D model and in 2D monolayer culture. Additionally, viability of GBM after drug treatment was higher when astrocytes and ECFCs were present in the 3D model compared to only GBM 3D culture and standard monolayer culture. Our results support the importance of the 3D-microenvironment as modulator of tumor response and provide a tunable in vitro platform for primary brain tumor studies aimed to compliment in vivo model observations.

Citation Format: Marisol Herrera-Perez, Melissa Fishel, Sherry Harbin, Jenna Rickus. Astrocytes and endothelial colony forming cells (ECFCs) influence the migration and drug response of glioblastoma cells in a 3D culture model. [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 613.
Abstract 63: A transposon-based forward genetic screen for steatosis-associated liver cancer drivers
Authors: Tschida Barbara R. , Temiz N. Alpay , Yang Ju D. , Keng Vincent W. , Largaespada David A.
Published: July 2016

Hepatocellular carcinoma (HCC), or liver cancer, is the second leading cause of death from cancer worldwide (Ferlay et al, Lyon, France: International Agency for Research on Cancer, 2013). HCC is linked with hepatic steatosis, or fatty liver disease, the most common chronic liver disease in the US. Both hepatic steatosis and HCC are increasing in prevalence in the US. However, the molecular mechanisms that cause HCC in a steatotic liver are not entirely understood. To uncover mutations promoting cancer in fatty livers and elucidate the role of steatosis in HCC, we performed a forward genetic screen using the conditional Sleeping Beauty (SB) transposon insertional mutagenesis system in mice treated with ethanol and a choline deficient diet to induce steatosis. Mice developed hepatic steatosis and liver cancer without sex bias, modeling the pattern seen in humans in whom HCC's with steatosis as a preceding condition are represented by a nearly equal number of males and females. We found that hepatic steatosis caused a downregulation of liver Foxa1 expression and increased Foxa2 inactivating phosphorylation, thus providing a molecular explanation for the loss of gender bias in our mouse model. Transposon insertion sites were mapped from the mouse HCC tumors, and genes near or in which more insertions were observed than expected by chance were identified as common insertion sites (CIS), which we consider putative steatosis-associated liver cancer genes. Wnt/b-catenin signaling, which defines a subclass of HCC and can induce tumors in mice without sex bias, was one of the top CIS associated pathways identified. Interestingly, alpha-adrenergic and dopamine signaling were also among the top steatosis-specific CIS associated pathways, suggesting a role for the sympathetic nervous system and cAMP signaling in fatty liver-associated cancer. Further studies are underway to test the roles of the pathways and individual genes identified in this study in driving HCC. Our studies implicate Wnt/beta-catenin and sympathetic nervous system signaling in steatosis-associated HCC and suggest methods for treatment or prevention in such cases.

Citation Format: Barbara R. Tschida, N. Alpay Temiz, Ju D. Yang, Vincent W. Keng, David A. Largaespada. A transposon-based forward genetic screen for steatosis-associated liver cancer drivers. [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 63.
Abstract 638: Clinical characteristics of breast cancer xenograft models
Authors: den Brok Wendie D. , Chia Stephen , Bates Cherie , Kalloger Steve , Aparicio Samuel , Mar Mar , Gelmon Karen , Eirew Peter
Published: July 2016

Purpose: We have expertise in breast cancer xenograft models and have previously published data on clonal dynamics. Our aim was to explore clinical characteristics of those patients (pts) whose breast cancer tumours engrafted versus those that did not.

Methods: Tissue from pts enrolled in a locally advanced/metastatic (MBC) study and a breast tumour tissue repository between Sept. 2008 and July 2014 underwent xenografting using NodScid/IL2rgKO (NSG) mice. Xenografts were passaged when tumour volume reached 1 cm3. Mice were sacrificed if no engraftment by 12 months (mos). Pt charts were reviewed to determine biomarker status (hormone receptor [HR], HER2), grade, LVI, time free from disease/progression, and pathologic complete response (pCR) for pts receiving neoadjuvant therapy.

Results: A total of 64 pts had known xenograft status: 32 engrafters, 32 non-engrafters. Biomarker status did not predict likelihood of engraftment (p = .0695) and is shown in Table 1 along with site/timing of biopsy for tissue engraftment within biomarker groups. When HER2+ cases are excluded from analysis, the HR-/HER2- phenotype yields a 72% probability of engraftment compared to 39% for the HR+/HER2- group (p = .0418). For engrafters, 22/32 (68%) of pts had or went on to have relapsed or de novo MBC. For non-engrafters, 7/32 (22%) had or went on to have relapsed disease (p = .0004). Grade and LVI did not predict engraftment (p = .1806 and p = .8657 respectively). No grade 1 tumours engrafted (n = 3). There were 25 pts who received neoadjuvant chemotherapy: 14 engrafters, 11 non-engrafters. None of the engrafters achieved a pCR; 3 non-engrafters achieved a pCR. Median time to engraftment was 5 mos for pts with relapsed/advanced disease vs 9.8 mos for pts who did not relapse however, treatment was variable.

Conclusion: This preliminary study highlights potential differences in clinical characteristics of engrafters vs non-engrafters in breast cancer xenograft models and warrants further exploration. (Funded by CBCRA, BCCF) TABLE 1.Biomarker status, tissue site/type in attempted xenografts.Engrafter (N = 32) N, (%)Non-engrafter (N = 32) N, (%)HR+/HER2-14 (44)22 (69)Tissue from:Primary tumour419Recurrence23Advanced dz on therapy80HR-/HER2-13 (40)5 (16)Tissue from:Primary tumour114Recurrence21Advanced dz on therapy00HER2+/HR+0 (0)2 (9)Tissue from:Primary tumour02Recurrence00Advanced dz on therapy00HER2+/HR-5 (16)3 (9)Tissue from:Primary tumour42Recurrence01Advanced dz on therapy10

Citation Format: Wendie D. den Brok, Stephen Chia, Cherie Bates, Steve Kalloger, Samuel Aparicio, Mar Mar, Karen Gelmon, Peter Eirew. Clinical characteristics of breast cancer xenograft models. [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 638.
Abstract 657: N-Myc and STAT Interactor knock out in the mammary epithelium prompts hyper-proliferation
Authors: Pruitt Hawley C. , Metge Brandon J. , Bailey Sarah K. , Shevde Lalita A. , Samant Rajeev S.
Published: July 2016

N-Myc and STAT Interactor (NMI) is an evolutionarily conserved protein that is widely expressed in fetal and adult tissues. Early studies implicated its role in regulating the activities of transcription factors (such as MYC, STATs, BRCA1, TIP60 etc.) critical to tumor progression and metastasis. However, the functional relevance of these regulatory activities of NMI remains unknown. Recent findings from our lab have revealed that NMI protein expression is decreased by 70% in primary tumor specimens from patients with metastatic breast cancer. Most recently, we have found that lack of NMI expression in breast cancer cells confers resistance to chemotherapy by blocking autophagy-induced cell death. Thus the status of NMI expression in breast cancer patients may be an important clinical consideration. Additionally, our functional studies have demonstrated that loss of NMI expression allows manifestation of TGFβ and Wnt driven EMT that results in increased invasion and metastatic dissemination. Overall, we have noticed a profound impact of NMI on multiple developmental signaling pathways that are essential for mammary development as well as tumor progression.

To further elucidate the biological role of NMI, we have created a genetically engineered mammary specific Nmi knock out model. We observed that in normal murine mammary tissue Nmi is expressed in the mammary epithelium during all stages of mammary development. However, it's expression is strikingly induced at the onset of pregnancy, implicating an important role of NMI in mammary ductal development and/or lactation. Remarkably, the Nmi knock out mice exhibit distinctly increased number of alveolar structures (30% more than in control mice) during lactation. Moreover, these Nmi-/- mammary glands also show 20% more proliferating mammary epithelial cells (MECs) compared to respective littermate controls. In addition, 3D-alveologenesis of Nmi knock out MECs is highly responsive to induction by growth factors such as TGFα and FGFs when compared to MECs from littermate controls. Hyper-proliferative phenotype has been implicated as one of the key factors for breast cancer initiation as well as progression. Overall, our work describes a direct evidence for the role of NMI as a key regulator of mammary epithelial cell proliferation.

Citation Format: Hawley C. Pruitt, Brandon J. Metge, Sarah K. Bailey, Lalita A. Shevde, Rajeev S. Samant. N-Myc and STAT Interactor knock out in the mammary epithelium prompts hyper-proliferation. [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 657.
Abstract 659: Novel refinements for the autochthonous mouse model of cancer
Authors: Cabeceiras Peter K. , Joshi Nikhil S. , Jacks Tyler
Published: July 2016

There are many levels of genetic regulation at play in autochthonous mouse models of cancer. For instance, mouse models can be programed to knockout tumor suppressor genes and enable transcription of oncogenes in order to cause normal cells to transform into cancer cells that can eventually form tumors and metastasize. Concurrently to this, a gene of interest (GOI) under the control of a tetracycline-response element (TRE) promoter can be studied in a doxycycline-dependent fashion. The GOI under the control of the TRE promoter will be transcribed only in the presence of a transcriptional activator fusion protein, rtTA, and a chemical compound, doxycycline. Unfortunately, it is extremely difficult to limit the doxycycline-dependent expression of the GOI only to cancer cells, so normal cells will invariably express the GOI. This inaccuracy can limit the translation of results from mouse model to human patient. In the interest of engineering models that closely recapitulate the disease in question, we propose to limit the expression of rtTA and the GOI to cancer cells by linking rtTA expression to cell cycle regulation. The cell cycle is leveraged because rapid division and proliferation are characteristics intrinsic to proliferative cancer cells, and some cell cycle proteins are abundantly expressed in cancer cells but not expressed in normal cells. By fusing rtTA to cell cycle proteins that are expressed in cycling cancer cells, we hope to improve the accuracy and capabilities of the autochthonous mouse model of cancer.

Citation Format: Peter K. Cabeceiras, Nikhil S. Joshi, Tyler Jacks. Novel refinements for the autochthonous mouse model of 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 659.
Abstract 667: Tumor cell dormancy is mediated by an increase in RTK signaling in reversible pancreatic cancer models expressing oncogenic K-Ras and c-Myc
Authors: Rajbhandari Nirakar
Published: July 2016

Recent genetic studies of pancreatic cancer expressing mutant Kras and c-Myc strongly favor targeted therapies against these oncogenic drivers for the successful treatment of advanced staged pancreatic cancer. The potential limitation of these treatments is that they likely leave residual cancer cells that can play a role in tumor recurrence. Our study is focused on understanding the molecular mechanism of the survival of pancreatic cancer cells in the absence of the tumor-driving oncogene in order to successfully eliminate cancer cells and prevent tumor relapse.

Using a reversible c-Myc-induced pancreatic cancer model, we have demonstrated that expression of c-Myc in pancreatic progenitors is sufficient to induce invasive pancreatic cancer, and its expression is required for the maintenance of tumors at primary and metastatic sites. However, despite the complete macroscopic remission of invasive pancreatic tumors at primary and metastatic sites upon downregulation of c-Myc, a few cancer cells survived and remained dormant for a protracted period of time (Lin et al., 2013). In this current study, we have used a reversible Kras-induced pancreatic tumor model and found that cancer cell dormancy is a common attribute in pancreatic cancer following oncogene ablation.

In an effort to identify common, cancer cell-intrinsic molecular pathways that mediate residual disease following the ablation of oncogenic drivers, we performed a genome-wide gene expression analysis (RNA-Seq) of in vivo-derived bulk tumor cells and dormant cancer cells that survived the ablation of c-Myc and oncogenic Kras. Comparison of the gene expression profile of bulk and residual cancer cells revealed that the residual cancer cells have increased receptor tyrosine kinase (RTK)/Akt signaling as a common mechanism for their survival in both c-Myc and Kras-induced tumors. We have experimentally validated the importance of this signaling pathway in cancer cell survival in the absence of oncogenic signaling in both tumor models. In comparison to control mice, we observed a significant slow tumor growth and delayed tumor relapse resulting in longer survival of the tumor bearing mice though a combination of this RTK/Akt pathway inhibition and ablation of Kras or c-Myc. In conclusion, our current study in reversible cancer models highlights the importance for the development of an adjuvant therapeutic strategy targeting this RTK/Akt pathway in addition to oncogenic drivers to effectively eradicate residual cancer cells and to prevent pancreatic cancer recurrence.

Citation Format: Nirakar Rajbhandari. Tumor cell dormancy is mediated by an increase in RTK signaling in reversible pancreatic cancer models expressing oncogenic K-Ras and c-Myc. [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 667.
Abstract 672: Differential effects of p53 mutations on cancer invasion and metastasis in a mouse model of colon cancer
Authors: Feng Ying , Sakamoto Naoya , Green Maranne , Greenn Megan , Cho Kathleen R. , Fearon Eric R.
Published: July 2016

Inactivation of the APC (adenomatous polyposis coli) tumor suppressor gene plays an important role in initiating most adenomas and colorectal cancers (CRCs). Somatic mutations in the TP53 tumor suppressor gene and KRAS oncogene are found in roughly 60% and 40% of CRCs, respectively, and contribute to tumor progression. In the vast majority of human CRCs, TP53 missense mutations lead to high levels of mutant p53 protein expression and the loss of the other wild type TP53 allele. To evaluate the role of TP53 missense mutations in CRC progression, we generated a genetically engineered mouse model of colorectal carcinoma, via combined targeting of Apc, Kras, and Trp53 alleles in mouse colon epithelium, focusing on comparing phenotypic effects of the murine equivalent of the human R273H mutation (i.e., murine R270H mutation) or a large deletion mutation of mouse Trp53. Inactivation of one allele Apc and activation of an oncogenic Kras allele in colon epithelium generated serrated and hyperplastic morphologic epithelium and adenomas. The addition of either the R270H missense mutation or the Trp53 null mutation to the Apc and Kras mutations led to markedly shortened survival of the mice, due to the development of multiple colon tumors in each mouse ranging from adenomas to late stage adenocarcinomas. Evidence of invasion into the smooth muscle and serosa area was found in both compound mice with missense or deletion mutations in Trp53, with the Trp53R270H mutant mice displaying an increased prevalence of deeply invasive tumors relative to the mice with deletion of Trp53. Furthermore, we found that the missense mutant Trp53R270H allele in combination with Apc and Kras mutations, but not the null-mutant Trp53 allele, was strongly linked to metastases to lymph nodes and lung. We have developed a useful mouse model of metastatic colon cancer that recapitulates the role of TP53 mutations in cooperating with APC and KRAS mutations in human CRC development and progression. In addition, our findings strongly suggest a powerful role for missense mutant p53 proteins compared to TP53 null mutations in promoting invasion and metastasis in CRC progression.

Citation Format: Ying Feng, Naoya Sakamoto, Maranne Green, Megan Greenn, Kathleen R. Cho, Eric R. Fearon. Differential effects of p53 mutations on cancer invasion and metastasis in a mouse model of colon 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 672.
Abstract 675: Ninjurin1, a target of p53, modulates p53-dependent tumor suppression in vivo
Authors: Yang Hee Jung , Cho Seong Jun , Zhang Jin , Yan Wensheng , Chen Xinbin
Published: July 2016

The nerve injury-induced protein 1 (Ninjurin1, Ninj1), a homophilic adhesion molecule, is involved in nerve regeneration, inflammation and vascular regression. Ninj1 is found to be overexpressed in human cancers including hepatocellular carcinoma and acute lymphoblastic leukemia. However, the role of Ninj1 in tumorigenesis has not been elucidated. Recently, we found that Ninj1 is transcriptionally regulated by p53, which in turn regulates p53 expression through mRNA translation. Thus, the mutual regulation between p53 and Ninj1 represents a novel loop in the p53 pathway. To examine the biological significance of the p53-Ninj1 loop, we generated and monitored 4 cohorts of mice: (i) WT, (ii) Ninj1-het, (iii) p53-null, (iv) Ninj1-het; p53-null. We found that Ninj1-het mice were highly prone to skin lesions. We also found that Ninj1 deficiency shortened the life-span but decreased the incidence of sarcoma in mice without p53. Additionally, we found that Ninj1-het; p53-null mice were prone to hydrocephalus and kidney abnormality. To examine the association of Ninj1 with mutant p53, we generated and characterized Ninj1+/-; p53 R270H/- and p53 R270H/- mice. We found that Ninj1 deficiency shortened the life-span and promoted the frequency and aggressiveness of tumors in p53 R270H/- mice. Together, we conclude that Ninj1 differently regulates the tumor susceptibility in a p53-dependent manner in vivo and that the Ninj1-p53 loop plays a pivotal role in tumor suppression and longevity.

Citation Format: Hee Jung Yang, Seong Jun Cho, Jin Zhang, Wensheng Yan, Xinbin Chen. Ninjurin1, a target of p53, modulates p53-dependent tumor suppression in vivo. [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 675.
Abstract 687: Critical role and mechanism of WASF3 in HER2/HER3 regulation of breast cancer metastasis
Authors: teng yong , Pi Wenhu , Cowell John
Published: July 2016

WASF3 is overexpressed in high-grade breast cancer and promotes invasion and metastasis but does not affect proliferation. The HER2/ERBB2/NEU gene is also frequently overexpressed in breast cancer and has been shown to promote invasion and metastasis in these tumors. Here we show that WASF3 in present in the HER2 immunocomplex and suppression of WASF3 function leads to suppression of invasion even in the presence of HER2 expression. Overexpression of both HER2 and WASF3 in non-metastatic MCF7 breast cancer cells promotes invasion and metastasis more significantly than either gene alone. HER2 forms homodimers as well as heterodimers with other HER family members and we now show that the ability of WASF3 to promote invasion is highly dependent on the HER2/HER3 heterodimer. The engagement of WASF3 with the HER2/HER3 complex facilitates its phospho-activation and transcriptional upregulation, which is facilitated by HER2/HER3 activation of JAK/STAT signaling. In breast cancer cells overexpressing HER2, therefore, WASF3 is specifically required to facilitate the invasion/metastasis response. Targeting WASF3, therefore, could be a potential therapeutic approach to suppress metastasis of HER2-overexpressing breast tumors.

Citation Format: yong teng, Wenhu Pi, John Cowell. Critical role and mechanism of WASF3 in HER2/HER3 regulation of 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 687.
Abstract 690: Functional characterization of RPS6KA3 (RSK2) and identification of its naturally occurring mutants in hepatocellular carcinoma
Authors: Chan Lo-Kong , Ho Daniel Wai-Hung , Chiu Yung-Tuen , Kai Alan Ka-Lun , Wong Chun-Ming , Ng. Irene Oi-Lin
Published: July 2016

RPS6KA3 encodes the p90 ribosomal S6 kinase 2 (p90RSK2 or RSK2), a protein kinase to relay the upstream transduction signal of the mitogen-activated protein kinase (MAPK) pathway. Currently, no characterization on RSK2 in human HCC has been performed. We determined the RSK2 mRNA expression in paired human HCC samples and their corresponding non-tumorous livers (n = 22) by real time PCR. RSK2 over-expression (≥2 folds) was found in 38% of the human HCCs and this over-expression was associated with tumor microsatellite formation, a feature of HCC metastasis. The oncogenic and pro-metastatic roles of RSK2 were further assessed and demonstrated in vivo by an orthotopic liver injection model in nude mice with stable shRSK2 knockdown HCC cells. Stable shRSK2 knockdown cells showed a significant reduction in tumor incidence in liver and lung metastasis. By exome-sequencing and targeted sequencing in a cohort of HBV-associated HCC patient samples (n = 111), we found that RSK2 was recurrently mutated in 6.3% of the samples being screened (n = 7). All the RSK2 mutants were confirmed by independent Sanger sequencing. Intriguingly, among the identified somatic RSK2 mutants, half of them introduced pre-mature stop gain to the transcript, which leads to the loss-of function of RSK2 through various mechanisms. Taken together, our data suggested that RSK2 may play differential roles in different subsets of HCC patients. Further characterization will definitely derive novel insights on the functional role of mutant RSK2 and its potential use to stratify HCC patients into different molecular subgroups.

(This study was supported by a RGC GRF fund (17116414), SK Yee Medical Research Fund 2011 and University Development Fund of The University of Hong Kong. Ng is Loke Yew Professor in Pathology)

Citation Format: Lo-Kong Chan, Daniel Wai-Hung Ho, Yung-Tuen Chiu, Alan Ka-Lun Kai, Chun-Ming Wong, Irene Oi-Lin Ng. Functional characterization of RPS6KA3 (RSK2) and identification of its naturally occurring mutants in hepatocellular 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 690.