bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5076: Tumor cell-derived exosomes educate bone marrow mesenchymal stromal cells toward a protumorigenic function
Authors: Nakata Rie , Borriello Lucia , Fabbri Muller , Shimada Hiroyuki , Declerck Yves A.
Published: August 2015

The bone marrow constitutes a microenvironment for tumor cells that promotes their survival, dormancy and therapeutic resistance. Central to this sanctuary function are mesenchymal stromal cells (MSC) which, like tumor-associated macrophages, can have an anti- or a pro-tumorigenic function in cancer progression. Here, using neuroblastoma, the second most common cancer in children that is highly metastatic to the bone marrow as a model of tumor cell - MSC communication - we have explored the mechanism by which tumor cells “educate” MSC toward a pro-tumorigenic function. We demonstrate that neuroblastoma cells release 30-100 nm size extracellular vesicles rich in tetraspanins (CD63) and chaperone proteins (HSP70), and therefore with characteristics of exosomes, and that they are captured by MSC. As a result, MSC increase their expression of several pro-tumorigenic cytokines and chemokines like interleukin (IL)-4, IL-6, IL-8, vascular endothelial cell growth factor (VEGF) and monocyte-chemotactic protein-1 (MCP-1). Capture of tumor-derived exosomes by MSC activates MEK/ERK1/2 and Akt (but not NF-κB), and whereas MEK/ERK1/2 activation is critical for the production of pro-tumorigenic cytokines/chemokines, Akt activation is not. In addition to the presence of structural, metabolic and chaperone proteins and the absence of inflammatory cytokines, a proteomic analysis of the cargo of these neuroblastoma-derived exosomes revealed an abundance of Galectin-3 binding protein (Gal-3BP), a protein that we had previously reported to increase the transcriptional expression of IL-6 in MSC. Further supporting the role of exosomes in educating MSC, we found that inhibition of exosome release in neuroblastoma cells by knock-down of Rab-27B, a GTPase involved in exosome secretion, was associated with a suppression of cytokine/chemokine production by MSC. We then demonstrated that “educated” MSC stimulate neuroblastoma cell proliferation both in vitro and in vivo and their resistance to chemotherapy in a signal transduction and activation (STAT)3 and MEK-ERK1/2- dependent manner. These data thus indicate that the production of exosomes by tumor cells induces the production of cytokines and chemokines in bone marrow-derived MSC, contributing to the protective microenvironment of the bone marrow niche.

Citation Format: Rie Nakata, Lucia Borriello, Muller Fabbri, Hiroyuki Shimada, Yves A. Declerck. Tumor cell-derived exosomes educate bone marrow mesenchymal stromal cells toward a protumorigenic function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5076. doi:10.1158/1538-7445.AM2015-5076
Abstract 5084: A multifaceted role of the Hedgehog pathway in modulating pancreatic ductal adenocarcinoma biology
Authors: Thomas Dafydd Huw , Lee Albert , Palermo Carmine , Sastra Stephen , Rabadan Raul , Olive Kenneth
Published: August 2015

The Hedgehog (Hh) developmental pathway is reactivated in pancreatic ductal adenocarcinoma (PDA) and forms a paracrine axis from the tumor cells to the supporting stromal cells. Previous work from this laboratory found that, in established tumors, inhibition of the Hedgehog (Hh) pathway caused a loss of tumor stroma implicating this axis in the development of the profuse desmoplastic stroma observed in PDA. However, the mechanisms by which Hh signaling modulates the stroma in PDA remain unknown.

In this study we have attempted to identify the target genes of the Hh pathway in the stroma of pancreatic tumors. Using a novel tumor biopsy procedure coupled with RNA-SEQ analysis we have identified genes acutely sensitive to Hh pathway inhibition in the KPC mouse model of PDA. The gene signature of down-regulated genes following Hh inhibition implicated the Hh pathway in the proliferation of its target stromal cells as well as a possible indirect effect on the proliferation of tumor cells. Interestingly, a significant number of genes were also up-regulated following inhibition of the Hh pathway. This gene signature was consistent with the migration and infiltration of leukocytes. Taken together these data suggest that the Hh pathway can drive proliferation of both the stromal and epithelial compartments as well as actively suppress leukocyte infiltration into pancreatic tumors.

In addition we have utilized a Gli1-GFP reporter mouse to study the Hh target cells in the stroma of KPC mice. Previous work has suggested that Hh target cells in pancreatic tumors are myofibroblasts and thus α-Smooth muscle actin (α-SMA) positive. However, we observed that while some Gli1 positive stromal cells are α-SMA positive a significant proportion are α-SMA negative. In addition, costaining KPC-Gli1-GFP tumors for other stromal markers, including Fibroblast specific protein-1 (FSP-1), Glial Fibrillary Acidic Protein (GFAP), Desmin, Vimentin and Platelet-derived growth factor receptor β (PDGFRβ) reveals subsets of Hh target cells in the tumor stroma. These data suggest the Hh pathway targets a complex set of stromal fibroblasts to mediate its effects.

Taken together, these data suggest that the Hh pathway regulates multiple facets of pancreatic tumor biology both in negative and positive manner and does so through targeting a complex array of stromal cells.

Citation Format: Dafydd Huw Thomas, Albert Lee, Carmine Palermo, Stephen Sastra, Raul Rabadan, Kenneth Olive. A multifaceted role of the Hedgehog pathway in modulating pancreatic ductal adenocarcinoma biology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5084. doi:10.1158/1538-7445.AM2015-5084
Abstract 5089: Desmoplastic stroma affects growth and invasion of progressively mutated human pancreatic cancer cells in vitro
Authors: Malik Ruchi , Luong Tiffany , Karamil Seda , Lelkes Peter , Cukierman Edna
Published: August 2015

Desmoplastic (desmo) stroma is a prognostic marker for pancreatic cancer, yet mechanisms behind its tumorigenic effects remain poorly understood. Tumor stroma, known to act permissive to naïve cells, may exacerbate tumor progression in mutated cells, whereas normal stroma acts restrictive to even aggressively mutated cells. The mechanistic details of these responses remain unclear. Previous studies suggested that a 3D microenvironment (i.e. Matrigel) is necessary for the manifestation of the metastatic phenotype in vitro in aggressively K-Ras mutated pancreatic cancer cells. Here we describe a new 3D in vitro model to study how human pancreatic stromal extracellular matrices (ECMs) interact with pancreatic epithelial cells, how these interactions activate specific integrins and downstream signaling pathways such as PI3K and ERK1/2, and how these signaling pathways may synergize with intrinsic mutational cues to enhance progression of pancreatic cancer. Our model is comprised of physiologically relevant desmo and normal 3D cell-derived matrices as well as polyacrylamide gel-based bioengineered ECMs. The influences of cell-derived and bioengineered ECMs were tested using a series of isogenic pancreatic ductal epithelial cells (PDEC) displaying different aggressive behavior.

In preliminary studies, we established that physiological desmo ECMs are tumor permissive (i.e. induce growth and/or invasion) for all PDEC cells (especially kRas mutated cells) when compared with matched restrictive/normal ECMs. In addition, K-Ras cells cultured on stromal activated (i.e. desmo) myofibroblast-derived 3D ECMs resulted in enhanced ERK1/2 activity, similar to what had been previously observed with Matrigel based system. In order to discern distinct cellular and signal responses mediated by cell-derived ECM in the various PDEC cells, we independently modulated microenvironmental cues such as stiffness, topography, and biochemical composition in the bioengineered ECMs. Our initial data suggests that the various PDEC cells differentially respond to alterations in microenvironmental cues. We anticipate that our studies will lead to better understanding of ECM-PDEC interactions in more detail, and help in identification of novel biomarker for early detection or prevention of pancreatic cancer.

Citation Format: Ruchi Malik, Tiffany Luong, Seda Karamil, Peter Lelkes, Edna Cukierman. Desmoplastic stroma affects growth and invasion of progressively mutated human pancreatic cancer cells in vitro. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5089. doi:10.1158/1538-7445.AM2015-5089
Abstract 509: MicroRNA-140 suppresses the migration and invasion of colorectal cancer cell possibly through targeting Smad3
Authors: Song Bo , Zhao Wenyue , Li Lianhong
Published: August 2015

Objective To investigate the roles of microRNA-140 (miR-140) on the migrating and invasive potentials of colorectal cancer (CRC) cell and the possible mechanism. Methods miR-140 mimics (miR-140), miR-140 specific inhibitor (Anti-miR-140) or siRNA against Smad3 (Smad3 siRNA) were transfected into human CRC cell line RKO respectively using Oligofectamine or Lipofectamine2000. Quantitative Real-time PCR (qRT-PCR) was used to measure the expression levels of miR-140 and Smad3 mRNA. Smad3 protein was analyzed by Western blot. The in vitro cell migrating and invasive potentials were determined by Wound-healing and Transwell chamber assays after up-regulating or down-regulating miR-140 or knocking down Smad3. Results The protein levels of Smad3 were significantly reduced after up-regulating miR-140 when compared to Control or Negative Control (NC) using Western blot. The results of qRT-PCR indicated that no significant difference was found in the levels of Smad3 mRNA between miR-140 transfection and NC (1.11±0.13 vs 1.00±0.06, P>0.05). Wound-healing assay showed that the migrating potential was dramatically attenuated by miR-140 compared to Control and NC, whereas no significance was found when compared to Smad3 siRNA transfecting cells. The cells migrating through Transwell chamber without matrigel in miR-140 group were 76.2±4.4, remarkably lowered than that in the Control (267.1±4.9) and NC (336.1±5.7), P values were all less than 0.05. There was no significant difference between miR-140 and Smad3 siRNA (83.5±7.3) groups. Transwell chamber with matrigel assay demonstrated that cells penetrating through the membrane were 109.5±7.4 in miR-140 group, dramatically lowered than that in the Control (403.1±5.1) and NC (392.6±8.4), P values were all less than 0.05. Smad3 siRNA transfection had the similar effect. Down-regulation of miR-140 increased the protein level of smad3, and partially reversed the inhibition of miR-140 on the potentials of cell migration and invasion. Co-transfection of miR-140 inhibitor and Smad3 siRNA had no significant effect on the Smad3 protein expression and the capacities of cell migration and invasion. Conclusions miR-140 directly targets Smad3 in the post-transcriptional level. miR-140 suppresses the migrating and invasive potentials of CRC cell, possibly through down-regulating Smad3. The findings of this study suggest that miR-140 may have a unique potential as a possible biomarker candidate for tumor metastasis diagnosis and therapy.

[Keywords] Colon neoplasms; microRNA-140; SMAD family member 3; Cell migration; Cell invasion

Citation Format: Bo Song, Wenyue Zhao, Lianhong Li. MicroRNA-140 suppresses the migration and invasion of colorectal cancer cell possibly through targeting Smad3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 509. doi:10.1158/1538-7445.AM2015-509
Abstract 5122: In vivo imaging models of bone and brain metastases and pleural carcinomatosis developed using a novel human EML4-ALK lung cancer cell line, A925LPE3
Authors: Nanjo Shigeki , Takeuchi Shinji , Kita Kenji , Fukuda Koji , Nakada Mitsutoshi , Nishihara Hiroshi , Yano Seiji
Published: August 2015

Introduction:EML4-ALK lung cancer accounts for approximately 3%-7% of non-small cell lung cancer cases. Crizotinib, an ALK tyrosine kinase inhibitor, shows dramatic clinical efficacy with a response rate of approximately 60%-80% in EML4-ALK NSCLC patients. However, almost all patients who strongly responded to crizotinib acquired resistance over time. Recently, new generation ALK-TKIs such as alectinib and ceritinib have been approved for treatment of ALK-rearranged NSCLC patients, and it is now clear that resistance may also develop against this class of inhibitors. To investigate the molecular mechanism underlying tumor progression and targeted drug sensitivity/resistance in EML4-ALK lung cancer, clinically relevant animal models are indispensable.

Methods: A human lung adenocarcinoma cell line A925L, which expresses an EML4-ALK gene fusion (variant 5a, E2:A20), established from a surgical specimen obtained from a Japanese male patient. We further established highly tumorigenic A925LPE3 cells with luciferase gene transfection, which also have the EML4-ALK gene fusion. A925LPE3 cells were injected into the right thoracic cavity for the pleural carcinomatosis model, into the tibia for the bone metastasis model, and into the right striatum for the brain metastasis model. After inoculation, the quantity of tumors and the quality of bones were tracked in live mice by repeated noninvasive optical imaging of tumor-specific luciferase activity using the IVIS Lumina XR Imaging System.

Results: In this study, we found that A925L and A925LPE3 cell lines are sensitive to the ALK inhibitors crizotinib and alectinib. By using A925LPE3 cells we established in vivo imaging models for pleural carcinomatosis, bone metastasis, and brain metastasis, all of which are significant clinical concerns of advanced EML4-ALK lung cancer. Interestingly, crizotinib caused tumors to shrink in the pleural carcinomatosis model, but not in bone and brain metastasis models, while alectinib showed remarkable efficacy in all three models, indicative of the clinical efficacy of these ALK inhibitors.

Conclusions: Our in vivo imaging models of multiple organ sites may provide useful resources to analyze further the pathogenesis of EML4-ALK lung cancer and its response and resistance to ALK inhibitors in various organ microenvironments.

Citation Format: Shigeki Nanjo, Shinji Takeuchi, Kenji Kita, Koji Fukuda, Mitsutoshi Nakada, Hiroshi Nishihara, Seiji Yano. In vivo imaging models of bone and brain metastases and pleural carcinomatosis developed using a novel human EML4-ALK lung cancer cell line, A925LPE3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5122. doi:10.1158/1538-7445.AM2015-5122
Abstract 5123: Curative fluorescence-guided surgery of pancreatic cancer in combination with UVC irradiation in orthotopic mouse models
Authors: Hiroshima Yukihiko , Maawy Ali , Zhang Yong , Sato Sho , Murakami Takashi , Yamamoto Mako , Uehara Fuminari , Miwa Shinji , Yano Shuya , Momiyama Masashi , Chishima Takashi , Tanaka Kuniya , Bouvet Michael , Endo Itaru , Hoffman Robert M.
Published: August 2015

Fluorescence-guided surgery (FGS) has shown great promise, but has not been shown to be curative. Human MiaPaCa-2 pancreatic cancer cells, expressing green fluorescent protein (GFP), were implanted orthotopically in nude mice. Brightlight surgery (BLS) was performed on 24 tumor-bearing mice. After BLS, mice were randomized to BLS-only (n = 8) or FGS (n = 8) or FGS-UVC (n = 8) groups. The residual tumors were resected using a portable imaging system. FGS or FGS followed by UVC irradiation with 2700 J/m2 UVC (254 nm) was performed. Residual tumor area after FGS was significantly smaller than after BLS only (p = 0.007). The BLS treated mice had reduced survival compared to FGS- and FGS-UVC-treated mice for both relapse-free survival (RFS) (p,0.001 and p,0.001, respectively) and overall survival (OS) (p,0.001 and p,0.001, respectively). With FGS-UVC, RFS lasted at least 150 days indicating the animals were cured. UVC irradiation in combination with FGS has curative potential in the clinic.

Citation Format: Yukihiko Hiroshima, Ali Maawy, Yong Zhang, Sho Sato, Takashi Murakami, Mako Yamamoto, Fuminari Uehara, Shinji Miwa, Shuya Yano, Masashi Momiyama, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Curative fluorescence-guided surgery of pancreatic cancer in combination with UVC irradiation in orthotopic mouse models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5123. doi:10.1158/1538-7445.AM2015-5123
Abstract 515: Cell fate determination factor DACH1 inhibits lung adenocarcinoma invasion and tumor growth through repression of CXCL5 signaling
Authors: Han Na , Yuan Xun , Wu Hua , Xu Hanxiao , Chu Qian , Guo Mingzhou , Yu Shiying , Chen Yuan , Wu Kongming
Published: August 2015

The onset of human lung cancer involves the loss of tumor suppressor genes and activation of tumor inducing genes. Current studies have proved EGFR, RAS, ALK as as key driver genes, however, in about 50% lung cancer samples there are no known genes to explain tumorigenesis, suggesting many novel pathways remain to be elucidated. Recent study on whole-genome and transcriptome sequencing NSCLC identified cell fate determinatiton gene DACH1 as novel alteration in genes involved in NSCLC. However, the role of DACH1 in NSCLC and its downstrem targets are largely unknown. We showed that expression of DACH1 was significantly decreased in human NSCLC tissues andits protein abundance was inversely correlated with the tumor stage and grade. Restoration of DACH1 function in NSCLC cells significantly reduced cellular proliferation, clone formation, migrasion and invasion in vitro, as well as tumor growth in vivo. By unbiased screen of secreting factors, we found out that CXCL5 was dramaticcaly repressed by DACH1. Furthermore, functional study suggested that CXCL5 was contribute to the DACH1-mediated repression of migrative in vitro and tumorigenesis in vivo. Consistingly, there was a inverse relationship between the mRNA level of DACH1 and CXCL5 in both lung cancer cell lines and huamn NSCLC tissues. Moreover, Kaplan-Meier analysis of human NSCLC samples demonstrated that high mRNA abundance of DACH1 was predicted favorate prognosis for both relaspse-free survivl and overall survival; in contrast, high CXCL5 expression predict worse prognosis for survival. To our understanding, this is the first study to demonstrate DACH1 regulated paracrine signaling in lung cancer and DACH1 could predict prognosis. In conclusion, DACH1 acts as a tumor suppressor gene through repression of paracrine signaling and is a potential biomarker for prognosis.

Citation Format: Na Han, Xun Yuan, Hua Wu, Hanxiao Xu, Qian Chu, Mingzhou Guo, Shiying Yu, Yuan Chen, Kongming Wu. Cell fate determination factor DACH1 inhibits lung adenocarcinoma invasion and tumor growth through repression of CXCL5 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 515. doi:10.1158/1538-7445.AM2015-515
Abstract 5152: Zebrafish models of myeloid malignancies produced through tet2 and asxl1 genomic editing
Authors: Gjini Evisa , Mansour Marc , Sander Jeffry , He Shuning , Ko Myunggon , Zhou Yi , Rodig Scott , Joung Keith , Zon Leonard , Rao Anjana , Look Thomas
Published: August 2015

Myelodysplastic syndromes (MDS) are one of the most common and most difficult to treat of all hematologic malignancies. MDS are diverse clonal disorders of hematopoietic stem cells in the bone marrow that cause dysplastic differentiation in developing progenitors leading to ineffective hematopoiesis, dangerously low circulating blood counts, and evolution to acute myeloid leukemia (AML). We sought to create a faithful zebrafish model of MDS, to study the altered molecular pathways and mechanisms underlying this disease and to provide a means to identify synthetic lethal relationships that can be exploited with small molecule inhibitors to selectively eliminate the mutant cells. Somatic loss-of-function mutations of the Ten-ElevenTranslocation-2 (TET2) and Additional Sex Combs Like 1 (ASXL1) genes are the most frequent clonal somatically acquired genetic abnormalities that have been identified in human MDS and other myeloid malignancies, such as chronic myelomonocytic leukemia (CMML). To generate a zebrafish model of MDS with tet2 and asxl1 loss, we used genome-editing technology to disrupt the zebrafish tet2 and asxl1 genes. The resultant tet2m/m mutant zebrafish are viable and fertile and exhibited normal embryonic and larval hematopoiesis, but developed myelodysplastic syndrome progressing to anemia at 24 months of age. asxl1 m/m mutant zebrafish are embryonic lethal with bone malformations. asxl1 m/m embryos show reduced numbers of hematopoietic and progenitor stem cells (HSPCs), a phenotype which is rescued by Tet2 loss. Combined loss of tet2 and asxl1 in the zebrafish leads to CMML at 5 months of age. These zebrafish lines provide animal models for small molecule and genetic screens in definitive HSPCs to identify synthetic lethal relationships and to dissect the pathways in hematopoietic cells that are altered by loss of function of tet2 and asxl1.

Citation Format: Evisa Gjini, Marc Mansour, Jeffry Sander, Shuning He, Myunggon Ko, Yi Zhou, Scott Rodig, Keith Joung, Leonard Zon, Anjana Rao, Thomas Look. Zebrafish models of myeloid malignancies produced through tet2 and asxl1 genomic editing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5152. doi:10.1158/1538-7445.AM2015-5152
Abstract 5170: The autotaxin-lysophosphatidic acid signaling pathways in ovarian carcinoma
Authors: Onalla Hadil , Tropé Claes , Reich Reuven , Davidson Ben
Published: August 2015

Ovarian carcinoma ranks as the fifth most common cancer among women. This is attributed to late presentation of the patients. The disease is characterized by widespread intraperitoneal metastases and accumulation of effusion fluids mainly in the peritoneal cavity

Lysophosphatidic acid (LPA) is a bioactive phospholipid with mitogenic and growth factor-like activities affecting cell invasion, cancer progression and resistance that act via specific cell-surface receptors present in many transformed cell types. LPA is produced by two main pathways, by PLA-2 and by Autotaxin (LysoPLD). Once its produced, it acts on six specific G protein coupled receptors, LPAR1-6. LPA has recently been implicated as a growth factor present in ascites of ovarian cancer patients. Previous studies have shown high levels of LPA in the effusions of ovarian carcinoma patients.

The aim of our study was to examine the role of ATX-LPA signaling in the progression of serous ovarian carcinoma. A total of 230 samples of serous ovarian carcinoma from the three different anatomic sites of the disease, were used .Effusion fluids were frozen and stored separately. All specimens were submitted for routine diagnostic purpose to the Division of Pathology at the Norwegian Radium Hospital during the period 1998-2008. mRNA levels of the six different LPA receptors were measured by RT-PCR, protein levels of Autotaxin were measured by Western blotting.

Results show that LPA2R is higher in effusion-derived tumor cells compared to solid lesions (p<0.001), while the opposite was found for LPA3R (p<0.001) and LPA6R (p<0.001). Clinico-pathological analysis showed that LPA3R levels are significantly higher in pre-chemo patients (p = 0.038). LPA2R (p = 0.021) and LPA5R (p = 0.035) were found higher in cases with larger residual disease volume. LPA5R (p = 0.032) was higher in cases with primary chemo-resistance and LPA1R (p = 0.05) was marginally associated with better overall survival.

Autotaxin protein levels were lower in effusion-derived tumor cells compared to solid lesions (p<0.001), yet higher levels of Autotaxin were found in peritoneal lesions compared to pleural ones (p = 0.033).

In this study we showed significant changes in mRNA LPAR levels and Autotaxin protein levels with the progression of the disease that correlates to clinic-pathological parameters.

To our best knowledge, this is the first description of the ATX-LPA signaling pathway in ovarian carcinoma with clinical relevance.

Citation Format: Hadil Onalla, Claes Tropé, Reuven Reich, Ben Davidson. The autotaxin-lysophosphatidic acid signaling pathways in ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5170. doi:10.1158/1538-7445.AM2015-5170
Abstract 5173: Androgen receptor status is highly conserved during tumor progression of breast cancer
Authors: Grogg André , Pfaltz Katrin , Lädrach Claudia , Cihoric Nikola , Zweifel Martin , Tapia Coya F. M.
Published: August 2015

Introduction: Many clinical trails are ongoing targeting androgen receptor (AR) in breast cancer (BC). Anti-androgen therapy is increasingly accepted in BC patients since the new compounds have less androgenic side effects. The assessment of AR status is generally performed on the primary tumor even when treating metastatic disease. To the best of our knowledge, nothing is known regarding discrepancies of AR status between the primary tumor and the metastatic site. Therefore, we investigated AR status on a large cohort of primary BCs and matched metastases.

Method: A next-generation Tissue Microarray (ngTMA) was constructed harboring 356 primary BCs, 133 matched metastases and 11 recurrences. A commercially available monoclonal AR antibody (DAKO) was used to evaluate AR-Status by immunohistochemistry. AR positivity was defined as ≥1% of nuclear staining in tumor cells. The results of the primary tumor and metastases/recurrences were compared. Additionally, the results of AR status were correlated with other pathological tumor features.

Result: AR could be evaluated in 353/356 (99.2%) of primary BCs and a positivity was detected in 307 (87%). A discordant result was seen in 4.3% metastasized BC's with informative results (5/117). Three negative BCs were positive in the metastases representing 16.7% of all negative BCs with informative results (3/18). Two BCs were positive in the primary and negative in the metastases representing 2.0% of all positive BCs with metastases and informative results (2/99). No discrepancies were seen between primary and loco-regional recurrences. Further, AR expression was significantly correlated with a positive estrogen (p<0.001) and progesterone receptor (p<0.001) status and a low proliferation (≤15%) (p<0.001). Negative AR status was significantly (p<0.001) associated with a higher tumor grade. Triple negative BCs were in 11.4% AR positive (4/35).

Conclusion: AR positivity is frequent in primary BCs (87%) and AR status is highly conserved during tumor progression. In a small fraction AR status changes (4.3%) either from positive to negative status or vise versa. However, even AR status is highly preserved we would recommend reevaluation of the metastatic site in negative BC's since a positive AR status can be seen in 16.7% of this subgroup.

Citation Format: André Grogg, Katrin Pfaltz, Claudia Lädrach, Nikola Cihoric, Martin Zweifel, Coya F. M. Tapia. Androgen receptor status is highly conserved during tumor progression of breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2015-5173
Abstract 518: O-GlcNAcylation regulates breast cancer cell invasion via the NAD+-dependent deacetylase SIRT1
Authors: Ferrer Christina M.
Published: August 2015

Cancer cells increase nutrient consumption leading to the altered metabolic state known as the Warburg effect. One pathway dependent on glucose, glutamine and acetyl-CoA is the Hexosamine Biosynthetic Pathway (HBP). Increased flux through the HBP leads to elevated post-translation addition of O-linked-β-N-acetylglucosamine (O-GlcNAc) on a diverse population of nuclear and cytosolic proteins, many of which are implicated in cancer. Recently, our lab provided the first evidence that breast and prostate cancers increases total O-GlcNAcylation by increasing O-GlcNAc Transferase (OGT) levels. Importantly, reducing OGT activity inhibits cancer cell invasion in vitro and metastasis in vivo. OGT inhibition reduces breast and prostate cancer cell invasion through, in part, inhibition of the oncogenic transcription factor, FOXM1 and its transcriptional target matrix metalloproteinase 2 (MMP2). Here, we show that OGT regulation of FOXM1 and cancer cell invasion requires regulation of the NAD+-dependent deacetylase, SIRT1. SIRT1 is a NAD+-dependent deacetylase regulated by changes in energy metabolism and its levels have been found to be reduced during breast cancer progression. We demonstrate an inverse relationship between O-GlcNAcylation and SIRT1 protein level and activity in multiple breast cancer cells and show that SIRT1 regulation by O-GlcNAcylation occurs in an AMPK-dependent manner. Consistent with this idea that reducing O-GlcNAcylation increases SIRT1 activity, we found that reducing OGT levels results in increased NAD+ metabolite levels in breast cancer cells. Importantly, we demonstrate that SIRT1 is critical for OGT-mediated regulation of FOXM1 protein ubiquitination and proteosomal degradation as reducing SIRT1 levels or activity reverses OGT-mediated regulation of FOXM1 and invasion in aggressive triple negative breast cancer cells. Furthermore, we show that SIRT1 regulates FOXM1 to modulate its transcriptional target MMP2 to promote breast cancer cell invasion. Collectively, these data indicate that flux through HBP, via O-GlcNAcylation, connects this nutrient sensing pathways with SIRT deactylases to regulate cellular invasion signaling via regulation of FOXM1.

Citation Format: Christina M. Ferrer. O-GlcNAcylation regulates breast cancer cell invasion via the NAD+-dependent deacetylase SIRT1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 518. doi:10.1158/1538-7445.AM2015-518
Abstract 5185: Identification of BRIP1 as a novel marker of breast tumor malignancy in the Omani population
Authors: Gupta Ishita , Ouhtit Allal , Al-Riyami Marwa , Al Ajmi Adil
Published: August 2015

The rationale of the present study is based on the following observations made in Oman: 1) a significantly increasing number of younger females (25-40 years) present to the clinic with advanced stage of breast cancer (BC); 2) the rate of consanguinity is significantly high (∼50%); and 3) the transition from normal/benign to malignant phenotype of breast tumor requires the involvement of a subset of specific genes. The long-term objective of this study was to identify and validate the gene(s) that underpin BC malignant transformation using functional genomic approaches. RNA samples were isolated from 40 malignant breast tumor tissues and 40 normal/benign breast tissues and analyzed by microarray gene expression profiling. Among a number of differentially expressed genes, BRIP1 showing a 5-fold up-regulation was identified as a potential gene that might underpin the transition from the benign to the malignant phenotype. The differential expression of BRIP1 was structurally validated by RT-PCR using the remaining RNA of the same samples previously examined by microarray. Pathway analysis was carried out to predict the major functional pathways involving BRIP1 to other identified genes. Ongoing sequencing of these genes using DNA extracted from the same samples will ultimately identify any genetic alteration that can affect the normal function of these genes. Functional validation assays will validate further the physiological relevance of BRIP1 in tumor malignancy, and perhaps other novel gene(s) specific to BC in the Omani population. This study has identified BRIP1 as a novel potential marker for BC malignancy, and can be a potential biomarker for early diagnosis of BC and/or a target to pave the way towards the design of anti-BC therapeutic strategies.

Citation Format: Ishita Gupta, Allal Ouhtit, Marwa Al-Riyami, Adil Al Ajmi. Identification of BRIP1 as a novel marker of breast tumor malignancy in the Omani population. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5185. doi:10.1158/1538-7445.AM2015-5185
Abstract 5211: In vivo fluorescence and spectral microscopy of the effects of aerobic exercise on tumor oxygenation and perfusion in breast cancer
Authors: Lee Jennifer A. , Wiggins Jennifer M. , Rice Lori P. , Siemann Dietmar W.
Published: August 2015

Abnormal tumor angiogenesis leads to inefficient blood flow and inhibited delivery of oxygen, resulting in a hypoxic tumor microenvironment. In radiation therapy the presence of oxygen is necessary to impart lasting damage to cancer cells, therefore the modulation of tumor hypoxia has been an important focus to improve radiation therapy outcome. While strategies such as hyperbaric chambers and hypoxic cell radiosensitizers or cytotoxins have shown merit, their success in the clinic has been limited. A potential method to increase oxygenation and decrease hypoxia in a tumor mass that is safe and well-tolerated is aerobic exercise. For cancer patients, the benefits of exercise in a palliative setting are well known, however the effects of exercise on tumor microvasculature and blood flow have not been extensively characterized.

In vivo microscopy of microvessel function in preclinical models is a useful tool for the study of tumor vascular structure and function. The combination of hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence (FPF) imaging of blood transit time in the murine dorsal skinfold window chamber has been previously used to characterize tumor angiogenesis, wound healing and the response of tumor microvasculature to vascular targeting agents. Hb saturation imaging reveals the oxygenation of blood within microvessels while FPF imaging gives information regarding blood flow and network connections by recording a fluorescent contrast agent as it is injected into circulation. In the present study, this combination imaging technique is used to evaluate the impact of treadmill running on tumor blood flow and microvessel oxygenation in real time. 4T1 mammary tumors were initiated in nude mice bearing titanium window chambers. Once tumors reached a volume of ∼5mm3, mice were subjected to daily treadmill running for 5-7 consecutive days (30 min, 15-18 m/min, 10° incline). As a sedentary control, mice were placed on a stationary treadmill for equivalent periods of time. Combination imaging was performed daily to monitor microvessel oxygenation and blood flow. On the first day of exercise, imaging was performed both before and after animals were exercised. Preliminary results indicate that following one bout of exercise, tumor perfusion is increased, however there was no significant correlation between exercise and tumor microvessel oxygenation.

Citation Format: Jennifer A. Lee, Jennifer M. Wiggins, Lori P. Rice, Dietmar W. Siemann. In vivo fluorescence and spectral microscopy of the effects of aerobic exercise on tumor oxygenation and perfusion in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5211. doi:10.1158/1538-7445.AM2015-5211
Abstract 5251: TaqMan® rare mutation assays for QuantStudio® 3D digital PCR system
Authors: Laig Marion , Ho Brian , Majumdar Nivedita S. , Lac Le T. , Chan Frances , Sathiyaa Ramesh , Russel Iain , Cifuentes Paco , Straub Ted , Varma Kamini , Keys David
Published: August 2015

Detection and quantification of mutant alleles in tumor tissue allow for research disease monitoring and the research of drug efficacy. Detection of emerging secondary mutations in the same tumor tissue causing resistance to potential treatment will help guide decisions on future treatment plans. A less invasive research method than using tumor tissue is testing for the presence of mutations in circulating free DNA (cfDNA).

We created a research tool for mutation detection at a sensitivity level of 1% and below. This allows researchers to find correlation between types of mutations and types of tumors and determination of potential secondary mutations.

The tool combines TaqMan® SNP Genotyping Assays with digital PCR. A set of assays was optimized for use in digital PCR with the QuantStudio® 3D Digital PCR System. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan® SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele.

Our initial set of 38 assays covers mutations commonly found in tumor tissues: BRAF V600E, mutations in EGFR exons 19, 20 and 21, KRAS codons 12 and 13, PIK3CA exons 9 and 20, and the JAK2 V617F mutations.

All assays were wet-lab tested at a 10% mutation rate and a 1% mutation rate using mutant plasmid spiked into wild-type genomic DNA. Additionally, selected assays were tested at the 0.1% mutation rate using mutant cell lines spiked into wild-type genomic DNA.

Wet-lab results confirm that all assays showed superior performance discriminating mutant and wild-type alleles. Mutant alleles were successfully detected as low as 0.1%.

For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Marion Laig, Brian Ho, Nivedita S. Majumdar, Le T. Lac, Frances Chan, Ramesh Sathiyaa, Iain Russel, Paco Cifuentes, Ted Straub, Kamini Varma, David Keys. TaqMan® rare mutation assays for QuantStudio® 3D digital PCR system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5251. doi:10.1158/1538-7445.AM2015-5251
Abstract 5255: miR-21 expression in HER2-positive breast cancer and prediction of resistance to adjuvant trastuzumab
Authors: Nielsen Boye S. , Balslev Eva , Poulsen Tim S. , Nielsen Dorte , Moller Trine S. , Mortensen Christiane E. , Holmstrom Kim S. , Hoegdall Estrid
Published: August 2015

Trastuzumab is established as standard care for patients with HER2-positive breast cancer both in the adjuvant and metastatic setting. However, approximately 50% of the patients do not respond to the trastuzumab therapy, and therefore new predictive biomarkers are highly warranted. MicroRNAs (miRs) constitute an interesting group of biomarkers and their cellular expression can be determined in tumor samples by in situ hybridization (ISH) analysis. miR-21 is highly prevalent and up-regulated in breast cancer and has been linked to drug resistance in clinical and in vitro settings. To determine expression patterns of miR-21 in high-grade breast cancers, we examined miR-21 expression in 22 HER2-positive tumors and 15 HER2-negative high-grade tumors by ISH. The histological examination indicated that patient samples could be divided into 3 major expression patterns: miR-21 predominantly in tumor stroma, predominantly in cancer cells or in both stromal and cancer cells. There was no obvious difference between the HER2-positive and HER2-negative tumors in terms of the miR-21 expression patterns and intensities. To explore the possibility that miR-21 expression levels and/or cellular localization could predict resistance to adjuvant trastuzumab in HER2-positive breast cancer patients, we analyzed additional 16 HER2-positive tumors from patients that were treated with trastuzumab in the adjuvant setting. Eight of the 16 patients showed clinical recurrence and were considered resistant. Examination of the miR-21 expression patterns and intensities revealed no association between the miR-21 scores in the cancer cell population (p = 0.69) or the stromal cells population (p = 0.13) and recurrent disease after adjuvant trastuzumab. Thus, our findings suggest that elevated miR-21 expression does not predict resistance to adjuvant trastuzumab.

Citation Format: Boye S. Nielsen, Eva Balslev, Tim S. Poulsen, Dorte Nielsen, Trine S. Moller, Christiane E. Mortensen, Kim S. Holmstrom, Estrid Hoegdall. miR-21 expression in HER2-positive breast cancer and prediction of resistance to adjuvant trastuzumab. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5255. doi:10.1158/1538-7445.AM2015-5255
Abstract 5273: Role of methylation of Wnt target genes in tumorigenesis and effect of re-expression with demethylating agent decitabine in colon cancer
Authors: Linnekamp Janneke , Kandimalla Raju , Vermeulen Louis , van Laarhoven Hanneke , Medema Jan Paul
Published: August 2015

Colon cancer is the fourth leading cause of cancer-related death worldwide. Extensive studies of the Wnt signal cascade have determined its role in colon cancer development and progress. Several targets of the Wnt-cascade like APCDD1, ASCL2, AXIN2 or DKK1 are thought to reduce Wnt activity by negative feedback. As a result, expression of these genes prevents pathway activation and silencing of these targets can cause proliferation of the tumor. Recently, we have shown that silencing of Wnt target genes by DNA-methylation predicts poor prognosis in colon cancer patients. Moreover, demethylation of those genes results in reexpression and is associated with reduced tumor growth in mice. Hence, treatment with demethylating agents can be beneficial for a subgroup of colon cancer patients with high methylation of Wnt target genes. The aim of this study is to clarify whether Wnt target gene methylation is an important driver in development of colon cancer and can be an effective target in colon cancer.

To determine the role of Wnt target genes in colon cancer more extensively, we study the effect of selective expression of these genes via an inducible vector system in established colorectal cell lines and in patient derived cultures that display CpG island methylation on these genes. In that way, we can monitor the effect of induced overexpression on tumor growth, tumor invasion and sensitivity towards chemotherapeutic agents. As a control, baseline expression in the same cell population, but in an uninduced state will be used.

Furthermore, we are also studying the effect of the demethylating agent decitabine on Wnt target gene methylation in a clinical trial in stage II/III colon cancer patients. Patients are pre-operatively treated with decitabine. Tumor material prior to decitabine treatment obtained during endoscopy is compared to resection material after treatment. Differences in methylation and expression of Wnt target genes are determined by respectively pyrosequencing and q-PCR.

We hypothesize that reexpression of Wnt target genes via an inducible vector results in lower Wnt pathway activity through negative feedback. As a result, tumor growth and invasion will be reduced and might be more sensitive towards chemotherapeutics.

To translate this into the clinic, demethylation of Wnt target genes by decitabine will hopefully give a similar effect in patients.

Citation Format: Janneke Linnekamp, Raju Kandimalla, Louis Vermeulen, Hanneke van Laarhoven, Jan Paul Medema. Role of methylation of Wnt target genes in tumorigenesis and effect of re-expression with demethylating agent decitabine in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5273. doi:10.1158/1538-7445.AM2015-5273
Abstract 5275: Association of TERT polymorphism with acute myeloid leukemia risk and prognosis
Authors: Lotfi Kourosh , Mosrati Mohamed Ali , Willander Kerstin , Jakobsen Falk Ingrid , Hermanson Monica , Höglund Martin , Stockelberg Dick , Wei Yuan , Söderkvist Peter
Published: August 2015

Telomerase reverse transcriptase gene (TERT) promoter mutations are identified in many malignancies but not in hematological malignancies. Here we analyzed TERT and protection of telomeres 1 gene (POT1) mutations, and four different TERT SNPs in 256 acute myeloid leukemia (AML) patients. A significant association for increased risk of AML was found for TERT SNPs, rs2853669 (OR = 2.45, p = 0.00006) and rs2736100 (OR = 1.52, p = 0.02). The overall survival for patients with CC genotype of rs2853669 was significantly shorter compared to those with TT or TC genotypes (p = 0.036 and 0.029 (respectively). The influence of TERT rs2853669 CC on survival was confirmed in multivariable Cox regression analysis as an independent risk biomarker in addition to high risk group, age and treatment. No hot spot TERT promoter mutations at -228C>T or -250C>T or POT1 mutations could be identified in this AML cohort. We show for the first time that rs2853669 CC may be a risk factor for the development of AML that may also be used as a prognostic marker to identify relative high risk NK-AML patients, for treatment guidance.

Note: This abstract was not presented at the meeting.

Citation Format: Kourosh Lotfi, Mohamed Ali Mosrati, Kerstin Willander, Ingrid Jakobsen Falk, Monica Hermanson, Martin Höglund, Dick Stockelberg, Yuan Wei, Peter Söderkvist. Association of TERT polymorphism with acute myeloid leukemia risk and prognosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5275. doi:10.1158/1538-7445.AM2015-5275