bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-103: L1CAM-targeted delivery of siRNA using elastin-like polypeptide (ELP) nanoparticles inhibits the growth of human tumors implanted in mice
Authors: Primiano Thomas , Chang Bey-Dih , Heidel Jeremy D.
Published: May 2015

Small interfering RNA (siRNA) drugs provide ideal means for perturbing intracellular oncogenic targets. However, specific delivery of siRNA to tumors has proven to be difficult. An ELP was engineered with an N-terminal region that binds to L1 cell adhesion molecule (L1CAM), and a C-terminal region that binds siRNA. Upon binding of siRNA, the L1CAM targeted ELP (ELP-L) spontaneously assembled into a spherical nanocomplex with the siRNA protected within, and the CAM-binding region protruding from the surface. These nanoparticles were used to deliver siRNA to SKOV3 tumors formed following surgical implantation of the cells into the left ovary of mice. Two weeks after implantation, mice were dosed with vehicle, ELP-L-complexed control siRNA (siCON), or ELP-L-complexed siEVI1, an siRNA to the oncogenic transcription factor EVI1, that is known to enable cell proliferation and inhibits apoptosis in ovarian tumor cells. To initially assess biodistribution, a single dose of ELP-L-DY800-labeled siEVI1 (10 mg/kg, ip) localized into ovarian tumors as early as 4 h, and remained for 72 h, as measured by co-registration of near infrared fluorescence with tumor bioluminescence in live mice. The ELP-L-DY800-siEVI1 was eliminated via kidney and did not accumulate in heart, lung, spleen or liver showing specific localization to tumors. To evaluate efficacy, the mean flux of bioluminescence from ovarian tumors of ten mice receiving 14 doses of 10 mg/kg siEVI1 in ELP-L over 28 days decreased by 92% with respect to mice dosed with 5% dextrose. Immunoblot and qPCR results showed EVI1 protein and mRNA levels decreased more than 50% in mice dosed with siEVI1 compared to siCON or vehicle. There were no observable adverse effects in the mice throughout the dosing period. These results are consistent with an RNA interference mechanism and suggest that ELP-L effectively delivers siEVI1 to tumors with limited toxicity. Furthermore, EVI1 appears to be a good target for ovarian cancer therapy and silencing by siRNAs is an effective means of triggering the death of ovarian tumor cells.

Citation Format: Thomas Primiano, Bey-Dih Chang, Jeremy D. Heidel. L1CAM-targeted delivery of siRNA using elastin-like polypeptide (ELP) nanoparticles inhibits the growth of human tumors implanted in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-103. doi:10.1158/1538-7445.AM2014-LB-103
Abstract LB-112: Highly potent and selective interleukin-1 receptor-associated kinase 4 inhibitors for the therapy of lymphoid malignancies
Authors: Kelly Priscilla N. , Chaudhary Divya , Young Ryan M. , Shaffer Art , Robinson Shaughnessy , Romero Donna L. , Kapeller Rosana , Staudt Louis M.
Published: May 2015

Pathologic activation of the Toll-like receptor (TLR) pathway underlies various human disorders such as autoimmune diseases, chronic inflammatory diseases and lymphoid malignancies. Current therapy of these diseases relies on immunosuppressive or chemotherapeutic agents, but more effective therapies tailored to disease-causing mechanisms are required. The IL-1 receptor-associated kinase 4 (IRAK4), is critical to TLR signaling and is recruited to TLRs by the adapter protein MyD88. Gain-of-function MYD88 mutations are activated by IRAK4 kinase in several mature B cell malignancies, including activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL).

Development of selective IRAK4 kinase inhibitors has been confounded by the challenging structure of IRAK4 catalytic domain. Using structure-based drug design methodologies, we identified potent and selective IRAK4 kinase inhibitors. ABC DLBCL cell lines that specifically harbor activating MYD88 mutations are killed by these inhibitors, both in vitro and in mouse xenograft models. Gene expression profiling revealed that IRAK4 kinase inhibitors downregulated prosurvival NF-κB signatures, and cytokine analysis showed a decrease in the production of inflammatory cytokines IL-6 and IL-10. Our findings open new possibilities for the therapy of malignant diseases that rely upon IRAK4 kinase.

Citation Format: Priscilla N. Kelly, Divya Chaudhary, Ryan M. Young, Art Shaffer, Shaughnessy Robinson, Donna L. Romero, Rosana Kapeller, Louis M. Staudt. Highly potent and selective interleukin-1 receptor-associated kinase 4 inhibitors for the therapy of lymphoid malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-112. doi:10.1158/1538-7445.AM2014-LB-112
Abstract LB-114: The long noncoding RNA HOTAIR promotes glioblastoma cell proliferation
Authors: Pastori Chiara , Penas Clara , Kapranov Philipp , St.Laurent Georges , Zhang Ming , Ayad Nagi , Wahlestedt Claes
Published: May 2015

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy in adults. The standard treatment consists of surgery followed by radiotherapy and chemotherapy, although this only modestly affects patient survival. Partial tumor resection and poor drug delivery to the brain impede effective treatment options. The development of high-throughput sequencing technology has allowed the cancer research community to identify hundreds of genetic alterations such as amplification, deletion, mutation, and changes in gene expression. However, all this information still needs to be translated into new therapeutic approaches.

These novel therapies may come from understanding long non-coding RNAs (lncRNAs). lncRNAs modulate transcription, regulate post-transcriptional RNA processing, translation, DNA methylation, and chromatin architecture through local (cis) and long distance (trans) mechanisms. Although relatively few lncRNAs have been functionally characterized, fewer are reported to have oncogenic or tumor suppressor properties. Recently, small signatures of 6 long-noncoding RNAs with altered expression have been reported for glioblastoma and these lncRNAs have shown correlation to overall survival.

Using the Helicos Next Generation Sequencing platform 12 GBM samples and 8 controls were sequenced. Our lab identified hundreds of lncRNAs potentially dysregulated in GBM. Among these there was the well-known lncRNA HOTAIR, which was found to be massively upregulated in GBM. This observation correlated with what has been observed in other cancers. However, increased expression of HOTAIR has been linked to poor prognosis due to metastatic events. Here we show that in glioblastoma HOTAIR does not promote metastasis, but instead sustains the ability of these cells to proliferate. We demonstrate that HOTAIR knockdown in GBM strongly impairs the proliferation of cells and induces apoptosis in vitro and in vivo experiments. To identify the direct gene targets of HOTAIR we employed the chirp technique (chromatin isolation by RNA precipitation), which allowed us to explain how this lncRNA affects gene transcription involved in proliferation/apoptosis and shed light on the cellular pathways regulated by HOTAIR. In addition to cell cycle regulation mediated by HOTAIR we find that certain epigenetic enzyme inhibitors potently downregulate HOTAIR expression. These exciting studies define a novel mechanism through which these enzymes control cell proliferation via lncRNA expression.

Citation Format: Chiara Pastori, Clara Penas, Philipp Kapranov, Georges St.Laurent, Ming Zhang, Nagi Ayad, Claes Wahlestedt. The long noncoding RNA HOTAIR promotes glioblastoma cell proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2014-LB-114
Abstract LB-115: Ultrasensitive detection of cancer mutations in metastatic colorectal cancer FFPE and cell-free DNA samples using multiplexed droplet digital PCR
Authors: Yang Wei , Shelton Dawne N. , Cooper Samantha , Berman Jennifer , Tzonev Svilen , Hefner Eli , Regan John F.
Published: May 2015

Targeted therapies in many forms of cancer today have allowed unprecedented progress in the treatment of disease. Despite these advances, routine implementation of genomic testing is still limited by: 1) methods to detect, with confidence, mutational loads below 10%, 2) limited amounts of sample (pg-ng range) per biological specimen, 3) diagnostic turnaround time, and 4) cost. In metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor antibodies (αEGFR) are used to target the wild-type EGFR receptor. However, KRAS is mutated in approximately 40% of colorectal cancers and is indicative of a negative response to αEGFR therapy. BRAF and PIK3CA mutations are also associated with poor response in the remaining patients with KRAS wild-type genotype. To optimize therapy strategies for personalized care, it is therefore critical to rapidly screen patient samples during the course of disease for the presence of multiple mutations.

The low abundance of mutants and limited amount and quality of available clinical samples (FFPE and cfDNA) render it difficult to reliably detect multiple mutations with current platforms and methods. We have developed a multiplexing strategy for screening clinically-actionable KRAS, BRAF, and PIK3CA mutations in mCRC clinical samples using digital PCR. No pre-amplification step was required. This sensitive and inexpensive method reduces the risk of contamination and can be easily implemented in molecular diagnostic laboratories for rapid, routine screening and monitoring of residual disease in cancer patients.

Citation Format: Wei Yang, Dawne N. Shelton, Samantha Cooper, Jennifer Berman, Svilen Tzonev, Eli Hefner, John F. Regan. Ultrasensitive detection of cancer mutations in metastatic colorectal cancer FFPE and cell-free DNA samples using multiplexed droplet digital PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-115. doi:10.1158/1538-7445.AM2014-LB-115
Abstract LB-12: EphA2 as a diagnostic imaging target in glioblastoma: A PET/MRI study
Authors: Puttick Simon , Stringer Brett W. , Day Bryan W. , Mardon Karine , Cowin Gary J. , Fay Michael , Thurecht Kristofer J. , Whittaker Andrew K. , Boyd Andrew W. , Rose Stephen
Published: May 2015

Neuroimaging has evolved from being a purely anatomical tool to a discipline that can offer anatomical, functional, hemodynamic, metabolic and molecular information. From the perspective of patients with glioblastoma multiforme (GBM), the use of neuroimaging techniques is an essential stage in diagnosis and classification of the tumour and to assess patient response following treatment.[1]

The Eph receptor tyrosine kinases (RTKs) and their Eph-interacting ligands (ephrins) are the largest family of RTKs consisting of 16 Eph receptors and 9 ephrin ligands.[2] From the discovery of the first Eph RTK evidence has been presented to implicate the Eph family in tumour development and progression, a body of work which continues to grow. The complex bidirectional signaling mechanisms between Eph RTKs and ephrin ligands have been shown to influence cell morphology, adhesion, migration, invasion, cell proliferation and survival.[2-4] Recently, many studies have linked Eph and ephrin expression levels with tumour progression and metastatic spread and, as a consequence, patient survival. In the specific case of EphA2, an increasing number of studies have shown a correlation between EphA2 overexpression and poor patient prognosis.[5,6] As such, EphA2 represents an attractive target for highly specific PET imaging using a labeled monoclonal antibody (mAb).

In this study we present simultaneous PET/MRI of a 64Cu labeled mAb specific to the human form of EphA2. We show efficient targeting of the tumour in three intracranial mouse models of GBM. In addition, we show that non-specific binding can be significantly reduced by blocking removal of the mAb from the blood pool via Fc receptor binding. In all cases we compare the ability of the mAb to delineate tumour boundaries with the current clinical imaging techniques of 18F-FDOPA PET imaging and gadolinium (Gd) contrast enhanced MRI.

[1] Henson, J. W.; Gonzalez, R. G. Expert review of neurotherapeutics 2004, 4.

[2] Pasquale, E. B. Nature Reviews Cancer 2010, 10, 165.

[3] Pasquale, E. B. Cell 2008, 133, 38.

[4] Pasquale, E. B. Nat Rev Mol Cell Bio 2005, 6, 462.

[5] Lackmann, M.; Boyd, A. W. Science Signaling 2008, 1.

[6] Zhuang, G. L. et al. Cancer Res 2010, 70, 299.

Citation Format: Simon Puttick, Brett W. Stringer, Bryan W. Day, Karine Mardon, Gary J. Cowin, Michael Fay, Kristofer J. Thurecht, Andrew K. Whittaker, Andrew W. Boyd, Stephen Rose. EphA2 as a diagnostic imaging target in glioblastoma: A PET/MRI study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-12. doi:10.1158/1538-7445.AM2014-LB-12
Abstract LB-123: Novel function of STAT1 pathway as a modulator of tumor progression in serous papillary endometrial cancer
Authors: Kharma Budiman , Baba Tsukasa , Matsumura Noriomi , Kang Hyun Sook , Murakami Ryusuke , Yamaguchi Ken , Hamanishi Junzo , Mandai Masaki , Murphy Susan K , Konishi Ikuo
Published: May 2015

Serous papillary endometrial cancers (SPEC) are highly progressive with poor prognosis, and its oncogenic profile is known different from endometrioid endometrial cancers. It, however, still remains unclear that which pathway promotes tumor progression. The IFN-γ transcription factor signal transducer and activator of transcription 1 (STAT1) has been considered as a tumor suppressor with transcription-dependent and transcription-independent mechanisms, but recent studies showed STAT1 was associated with poor prognosis in some cancers with unclear mechanisms.

In this study, we performed a genome-wide analysis by microarray analysis of 63 human endometrial cancers which revealed STAT1 pathway was constitutively up-regulated in SPEC, and its expression was confirmed significantly higher in SPECs tissue cancer by immunohistochemical staining (p<0.001). Immunohistochemical staining also exhibited co-localization of ICAM-1 and PD-L1 at tumor frontier with CD8-T cells (p<0.001) as well as STAT1 expression in SPECs. Using a SPEC cell line, SPAC-1L, it was confirmed that IFN-γ induced STAT1 expression not only to promote cellular proliferation (p<0.05), adhesion (p<0.0001), and invasion (p=0.0002), but to induce expression of cMyc, ICAM-1 and PD-L1 (p<0.05). In contrast, suppression of STAT1 attenuated induction of these genes (p<0.05) and inhibited xenograft tumor growth on NOD-SCID mice (p<0.0001).

These results indicate that STAT1 pathway modulates the micro-environment of SPECs in driving its downstream genes to promote tumor progression. Furthermore, it could be developed as a novel potential target for molecular therapy in SPEC.

Citation Format: Budiman Kharma, Tsukasa Baba, Noriomi Matsumura, Hyun Sook Kang, Ryusuke Murakami, Ken Yamaguchi, Junzo Hamanishi, Masaki Mandai, Susan K Murphy, Ikuo Konishi. Novel function of STAT1 pathway as a modulator of tumor progression in serous papillary endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-123. doi:10.1158/1538-7445.AM2014-LB-123
Abstract LB-126: ARID1B is a specific vulnerability in ARID1A-mutant cancers
Authors: Helming Katherine , Wang Xiaofeng , Wilson Boris , Vazquez Francisca , Haswell Jeffrey , Manchester Haley , Kim Youngha , Kryukov Gregory V. , Ghandi Mahmoud , Aguirre Andrew J. , Jagani Zainab , Wang Zhong , Garraway Levi , Hahn William , Roberts Charles
Published: May 2015

Cancer genome sequencing efforts have revealed that ARID1A is frequently mutated in many types of cancer. Inactivating mutations occur in 45% of ovarian clear cell and endometrioid carcinomas, 19% of gastric cancers, 19% of bladder cancers, 14% of hepatocellular cancers, 12% of melanomas, and less frequently in colorectal, lung, breast and pancreatic cancers. ARID1A has also been validated as having bona fide tumor suppressor properties. Consequently, identification of specific vulnerabilities conferred by ARID1A mutation would have major relevance for human cancer. We therefore performed a systematic search for genetic vulnerabilities created by ARID1A mutation. Out of 9000+ genes screened, ARID1B, a related but mutually exclusive homolog of ARID1A in the SWI/SNF chromatin remodeling complex, was the number one gene preferentially required in ARID1A-mutant cancer cells. In validation studies, we find that knockdown of ARID1B indeed specifically impairs the proliferation of ARID1A-mutant cancer cell lines. Mechanistically, we show that loss of ARID1B in ARID1A-mutant cancer cell lines destabilizes the SWI/SNF complex and results in dissociation of the catalytic ATPase subunit SMARCA4. We further show that this finding also holds true in primary murine fibroblasts genetically engineered conditional for ARID1A. Our findings demonstrate that cancer cell lines harboring mutations in ARID1A are specifically dependent upon ARID1B and provide insight into the structural role of ARID1A/B in the SWI/SNF complex. Intriguingly, we also find that ARID1A and ARID1B are frequently co-mutated in cancer, but that all ARID1A-deficient cancers retain at least one ARID1B allele. These results suggest that loss of ARID1A and ARID1B alleles cooperatively promotes cancer formation but also results in a unique functional dependence. These results also indicate that ARID1B could be considered a potential therapeutic target for the wide variety of ARID1A-mutant human cancers.

[K.H. and X.W contributed equally to this work.]

Citation Format: Katherine Helming, Xiaofeng Wang, Boris Wilson, Francisca Vazquez, Jeffrey Haswell, Haley Manchester, Youngha Kim, Gregory V. Kryukov, Mahmoud Ghandi, Andrew J. Aguirre, Zainab Jagani, Zhong Wang, Levi Garraway, William Hahn, Charles Roberts. ARID1B is a specific vulnerability in ARID1A-mutant cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-126. doi:10.1158/1538-7445.AM2014-LB-126
Abstract LB-131: Elevated (L) -2-hydroxyglutarate promotes loss of 5-hydroxymethylcytosine in clear cell renal cancer
Authors: Shim Eun-Hee , Livi Carolina B. , Knight John , Holmes Ross P. , Rakheja Dinesh , Velu Sadanan , Kho Eun-Young , Chenna Balachandra , Rea Shane L. , Benson Daniel , Kirkman Richard , Ghosh Arindam , Li Qiuhua , Bae Sejong , Wei Shi , Block Karen L. , Sudarshan Sunil
Published: May 2015

We undertook an unbiased metabolomics profile of clear cell renal cell carcinoma (ccRCC), the most common histological subtype, to gain deeper insight into the metabolic basis of this disease. Here, we demonstrate elevations of the metabolite 2-hydroxyglutarate (2HG) in kidney cancer. Elevation of 2HG, specifically the D- enantiomer, has been linked to transformation and identified in the context of gain of function mutations of isocitrate dehydrogenase (IDH1 and IDH2). Notably, enantiomeric resolution demonstrates that, in contrast to IDH1/IDH2 mutant tumors, kidney tumors demonstrate elevations of the L enantiomer of 2HG (L-2HG). Moreover, high 2HG tumors demonstrate reduced DNA levels of 5-hydroxymethylcytosine (5hmC) consistent with the ability of L-2HG to inhibit TET (Ten Eleven Translocation) enzymes which convert 5-methylcystoine to 5-hmC. Finally, we demonstrate that these changes are mediated by the reduced expression of L-2HG dehydrogenase (L2HGDH) in ccRCC.

Here, we report elevations of the putative oncometabolite L-2HG in the most common subtype of kidney cancer and describe a novel mechanism for the regulation of DNA 5hmC levels. Our findings provide new insight into the metabolic basis for the epigenetic landscape of renal cancer.

Citation Format: Eun-Hee Shim, Carolina B. Livi, John Knight, Ross P. Holmes, Dinesh Rakheja, Sadanan Velu, Eun-Young Kho, Balachandra Chenna, Shane L. Rea, Daniel Benson, Richard Kirkman, Arindam Ghosh, Qiuhua Li, Sejong Bae, Shi Wei, Karen L. Block, Sunil Sudarshan. Elevated (L) -2-hydroxyglutarate promotes loss of 5-hydroxymethylcytosine in clear cell renal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2014-LB-131
Abstract LB-133: Orchestrated recruitment of histone methyltransferases to DNA double strand breaks facilitates 53BP1 binding and proficient repair
Authors: Tuzon Creighton T. , Spektor Tanya M. , Congdon Lauren M. , Kong Xiaodong , Cheung Peggie , Kuo Alex J. , Yokomori Kyoko , Gozani Or , Rice Judd C.
Published: May 2015

While selective binding of the tandem tudor domains of p53 binding protein 1 (53BP1) to dimethylated histone H4 lysine 20 (H4K20me2) at DNA double strand breaks (DSBs) is well-documented to be an essential event for proficient DSB repair, the enzyme(s) responsible for H4K20 methylation at DSBs remained unknown. Contrary to previous reports implicating the MMSET/NSD2 methyltransferase in this pathway, we demonstrate that MMSET is dispensable for H4K20 methylation, 53BP1 accumulation and DSB repair. Alternatively, we discovered that the PR-Set7 H4K20 monomethyltransferase (H4K20me1) is an essential component of nonhomologous end joining (NHEJ) which functions early and directly in DSB repair. PR-Set7 was found to mobilize immediately to DSBs and PR-Set7 enzymatic activity was necessary for H4K20 methylation, 53BP1 accumulation and proficient DSB repair. Identification of PR-Set7-associated proteins revealed interactions between PR-Set7 and several NHEJ repair proteins. Consistent with these findings, the rapid recruitment of PR-Set7 to induced DSBs was dependent on direct binding to the NHEJ Ku70/Ku86 heterodimer. PR-Set7-mediated H4K20me1 was found to be necessary, but not sufficient, for H4K20me2 and 53BP1 recruitment at induced DSBs and an undamaged ectopic locus. In addition, we also discovered that the Suv4-20 H4K20 dimethyltransferases mobilize to DSBs and were required for H4K20me2 and 53BP1 accumulation. Importantly, Suv4-20 was not sufficient for H4K20me2 and 53BP1 localization at induced DSBs or an undamaged ectopic locus in cells lacking a catalytically competent PR-Set7. Therefore, these results illuminate an orchestrated molecular pathway whereby PR-Set7-mediated H4K20me1 “primes” the DSB to expedite subsequent catalysis by Suv4-20 for the generation of H4K20me2 at the lesion necessary for 53BP1 binding. The collective findings identify PR-Set7 as an essential early component of NHEJ and reveal a novel temporal PR-Set7-dependent pathway that facilitates H4K20me2 and 53BP1 accumulation at DSBs necessary for proficient DSB repair. Furthermore, these results and preliminary studies indicate that small molecule inhibitors targeting PR-Set7 and/or Suv4-20 will likely be clinically effective radio- and chemo-sensitizing agents in cancer treatment.

Citation Format: Creighton T. Tuzon, Tanya M. Spektor, Lauren M. Congdon, Xiaodong Kong, Peggie Cheung, Alex J. Kuo, Kyoko Yokomori, Or Gozani, Judd C. Rice. Orchestrated recruitment of histone methyltransferases to DNA double strand breaks facilitates 53BP1 binding and proficient repair. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-133. doi:10.1158/1538-7445.AM2014-LB-133
Abstract LB-145: Astrocytes directly influence tumor cell invasion and metastasis in vivo
Authors: Wang Ling , Cossette Stephanie , Rarick Kevin , Gershan Jill , Michael Michael , Harder David , Ramchandran Ramani
Published: May 2015

Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among which, tumor invasion factors namely matrix metalloprotease-2 (MMP-2) and MMP-9 were partly responsible for the astrocyte media-induced tumor cell invasion. Inhibiting MMPs reduced the ability of tumor cell to migrate and invade in vitro. Further, injection of astrocyte media-conditioned breast cancer cells in mice showed increased invasive activity to the brain and other distant sites. More importantly, blocking the preconditioned tumor cells with broad spectrum MMP inhibitor decreased the invasion and metastasis of the tumor cells, in particular to the brain in vivo. Collectively, our data implicate astrocyte-derived MMP-2 and MMP-9 as critical players that facilitate tumor cell migration and invasion leading to brain metastasis.

Citation Format: Ling Wang, Stephanie Cossette, Kevin Rarick, Jill Gershan, Michael Michael, David Harder, Ramani Ramchandran. Astrocytes directly influence tumor cell invasion and metastasis in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-145. doi:10.1158/1538-7445.AM2014-LB-145
Abstract LB-148: Inflammatory cytokines alter the sensitivity of breast cancer cells to endocrine treatments
Authors: Stender Joshua D. , Kastrati Irida , Yakir Maayan , Frasor Jonna , Glass Christopher K.
Published: May 2015

In breast cancer, infiltration of macrophages establishes an inflammatory tumor microenvironment, which can foster an extremely aggressive and therapy resistant tumor. Although inflammatory cytokines are known to modulate the transcriptional potential of estrogens and contribute to an endocrine-resistant state in cell-based model systems, the underlying molecular mechanisms remain poorly defined. We have used the human breast cancer cell-line MCF-7, an estrogen receptor α(ERα) positive and inflammatory cytokine sensitive cell line, to define the global impact inflammatory cytokines on the ERα cistrome and ERα-dependent transcriptional activity using next-generation sequencing methods. IL-1β and TNFα treatments establish an ERα cistrome that substantially overlaps with the E2-dependent ERα cistrome and results in ERα-dependent, ligand-independent activation of gene expression. Cytokine stimulation of MCF-7 cells results in phosphorylation of ERα at S305 which drives the ligand-independent activation of ERα. Pharmacological inhibition of IKKα/β blocks cytokine-dependent S305 phosphorylation, inhibits the cytokine-dependent ERα cistome, and abolishes cytokine-dependent gene activation. Additionally, S305 phosphorylation by cytokine treatments blocks the ability of tamoxifen to inhibit the expression of a subset of E2-dependent target genes and mutation of S305 to alanine restores tamoxifen sensitivity in the presence of IL-1β or TNFα. Collectively, these results provide molecular details dictating how inflammatory cytokines activate the transcriptional potential of ERα and drive an endocrine-resistant state in human breast cancer cells. In addition, they highlight the potential for anti-inflammatory therapies to be utilized in the treatment of patients with tamoxifen-resistant breast cancer.

Citation Format: Joshua D. Stender, Irida Kastrati, Maayan Yakir, Jonna Frasor, Christopher K. Glass. Inflammatory cytokines alter the sensitivity of breast cancer cells to endocrine treatments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-148. doi:10.1158/1538-7445.AM2014-LB-148
Abstract LB-153: Influence of macrophages and p38α/β signaling on early metastatic dissemination of premalignant ErbB2+ mammary epithelial cells
Authors: Linde Nina , Mortha Arthur , Sosa Soledad , Merad Miriam , Aguirre-Ghiso Julio
Published: May 2015

Metastases originate from tumor cells that disseminate from the primary site through a process aided by macrophages. This was proposed to occur only during late invasive stages of cancer. However, the presence of tumor cells in the bone marrow of patients with ductal carcinoma in situ (DCIS), a pre-invasive form of breast cancer, argues that these events can take place earlier. The mechanisms driving this process are poorly understood. Our lab showed that in the MMTV-ErbB2 breast cancer model, inhibition of p38 signaling accelerated disease progression and enhances early dissemination. Using this same model and knowing that macrophages are key for mammary gland development and remodeling, we investigated whether these inflammatory cells might actively participate in early dissemination. We now show that F4/80+ macrophages associate with mammary ducts in the periphery of the myoepithelial layer but are never inside the ducts in wild type or in pre-malignant 18-20 week old MMTV-ErbB2 mice mammary glands. We found that macrophages are recruited into epithelial layers of pre-malignant lesions, when the ErbB2 oncogene is amplified and p38 activity is lost. This event showed a high correlation with enhanced dissemination of ErbB2+CK8/18+ mammary epithelial cells (MECs) detected in circulation or the bone marrow. Sites where macrophages had invaded the ducts showed disruption of the myoepithelium and reduced E-Cadherin expression in MECs. Depletion of macrophages from pre-malignant MMTV-Neu mice abolished p38 driven early dissemination. Importantly, DCIS samples from patients frequently contained intra-epithelial macrophages. These data suggest that macrophages may actively aid early dissemination from pre-malignant tissue, but only in the context of p38 inhibition and ErbB2 activation. Further, in areas of macrophage infiltration, luminal ductal cells may downregulate E-cadherin, which could foster cell migration/invasion. Our data in human DCIS samples further underscores the relevance of our findings and indicates that analysis of macrophages in DCIS lesions might be useful to better identify patients at high risk of carrying disseminated disease.

Citation Format: Nina Linde, Arthur Mortha, Soledad Sosa, Miriam Merad, Julio Aguirre-Ghiso. Influence of macrophages and p38α/β signaling on early metastatic dissemination of premalignant ErbB2+ mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-153. doi:10.1158/1538-7445.AM2014-LB-153
Abstract LB-159: Characterization of cell-cycle progression using a novel bi-cistronic lentiviral FUCCI reporter
Authors: Pineda Gabriel , Lambert-Fliszar Florence , Riso Gennarina L. , Kane Kathleen M. , Jamieson Catriona
Published: May 2015

Currently, there are few methods to visualize cell cycle progression in leukemia stem cells (LSC). Human LSC are characterized as quiescent self-renewing cells that drive leukemic transformation of myeloproliferative neoplasms and myelodysplastic syndromes. Additionally, current methods require sequential transduction and clonal selection. These methods and conditions are not feasible to study primary patient samples and LSC, which are generally limited in sample size. To alleviate this challenge, we generated a lentiviral biscistronic vector encoding FUCCI (fluorescent ubiquitination-based cell-cycle indicator) probes. Time-lapse confocal imaging of transduced 293 cells and SKNO-1 cells exhibited normal cell division and distinct nuclear staining of either green or red fluorescence. Further, analysis by flow cytometry of 293 cell confirms that S/G2/M and G1/G0 phases of cell cycle correlate to expression of fluorescent reporters expressed from bicistronic lentiviral vector and recapitulate FACS plots using traditional Ki67 and 7AAD staining. The development of this new reagent offers a new and more effective method to track cell cycle progression in limited mammalian cell populations. Future studies will utilize primary patient CD34+CD38- selected leukemic stem cell populations.

Citation Format: Gabriel Pineda, Florence Lambert-Fliszar, Gennarina L. Riso, Kathleen M. Kane, Catriona Jamieson. Characterization of cell-cycle progression using a novel bi-cistronic lentiviral FUCCI reporter. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-159. doi:10.1158/1538-7445.AM2014-LB-159
Abstract LB-162: Translational imaging of tumorigenesis in a TP53 porcine cancer model
Authors: Sieren Jessica C. , Wang Xiao-Jun , Davis Bryan , Newell John D. , Hammond Emily , Rohret Judy , Rohret Frank , Struzynski Jason , Goeken Adam , Naumann Paul , Leidinger Mariah , Hagen Jussara , Rheeden Richard Van , Darbro Benjamin W. , Quelle Dawn E. , Meyerholz David K. , Rogers Christopher S.
Published: May 2015

Cancer remains the second deadliest disease in the United States, necessitating improvements in tumor diagnosis and treatment. Current model systems of cancer have been informative but also present challenges to translating promising imaging approaches and therapies to the clinic. The lack of a large animal model that accurately mimics human cancer has been a major barrier to the development of effective diagnostic tools and interventions (surgical and therapeutic). This study sought to 1) develop a genetically-modified porcine model of cancer expressing a TP53 mutation commonly found in humans (R175H in people, R167H in pigs), 2) validate the cellular p53 mutation and 3) evaluate tumor development in the animals using medical imaging, histopathology and molecular approaches. TP53R167H/R167H mutant pigs primarily developed lymphomas and osteogenic tumors, mimicking the tumor types observed in mice and humans expressing the orthologous TP53 mutant allele. CT and MRI imaging data effectively detected developing tumors, which were validated by histopathologic evaluation following necropsy. Molecular genetic analyses confirmed mutant p53 expression and characteristic chromosomal instability within the tumors. In conclusion, TP53R167H/R167H pigs provide a novel large animal tumor model that replicates the human condition and is uniquely suited to developing clinically-relevant, non-invasive imaging approaches to facilitate earlier detection, diagnosis and treatment of human cancers.

Citation Format: Jessica C. Sieren, Xiao-Jun Wang, Bryan Davis, John D. Newell, Emily Hammond, Judy Rohret, Frank Rohret, Jason Struzynski, Adam Goeken, Paul Naumann, Mariah Leidinger, Jussara Hagen, Richard Van Rheeden, Benjamin W. Darbro, Dawn E. Quelle, David K. Meyerholz, Christopher S. Rogers. Translational imaging of tumorigenesis in a TP53 porcine cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-162. doi:10.1158/1538-7445.AM2014-LB-162
Abstract LB-167: Tumor cells, but not endothelial cells, mediate the eradication of primary cancers by radiation therapy
Authors: Moding Everett J. , Lee Chang-Lung , Castle Katherine D. , Oh Patrick , Kirsch David G.
Published: May 2015

Radiation therapy is frequently utilized in the clinic as curative treatment for cancers, but the contribution of endothelial cells to tumor response to radiation remains controversial. Using the two highly efficient site-specific recombinases, Cre and FlpO, we have generated primary tumors in mice with different gene mutations specifically in tumor cells and stromal cells. With this dual recombinase technology, we selectively mutated the proapoptotic gene Bax or the DNA damage response gene Atm to genetically manipulate the radiosensitivity of endothelial cells in primary soft tissue sarcomas. We found that deletion of Bax in endothelial cells did not affect tumor response to radiation, but Atm deletion increased radiation-induced death of tumor endothelial cells and prolonged tumor growth delay following a non-curative dose of radiation. However, following a curative dose of radiation, Atm deletion in endothelial cells did not affect growth delay of primary tumors and failed to increase local control. In contrast, deletion of Atm in tumor cells increased local control of primary tumors by radiation therapy. These results demonstrate that tumor cells rather than endothelial cells are the critical targets that regulate tumor eradication by radiation therapy. They also emphasize the importance of using primary models of cancer to study the role of stromal cells in tumor development and response to therapy.

Citation Format: Everett J. Moding, Chang-Lung Lee, Katherine D. Castle, Patrick Oh, David G. Kirsch. Tumor cells, but not endothelial cells, mediate the eradication of primary cancers by radiation therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-167. doi:10.1158/1538-7445.AM2014-LB-167
Abstract LB-174: MicroRNA-126 (miR-126) and epidermal growth factor-like domain 7 (EGFL7) in Bevacizumab-treated patients with metastatic colorectal cancer
Authors: Nielsen Boye S. , Sorensen Flemming B. , Johnsson Anders , Jakobsen Anders , Hansen Torben F.
Published: May 2015

The miR-126 gene is located within intron 7 of the EGFL7 encoding gene, and therefore miR-126 expression is dependent on the transcription of the EGFL7 gene. It has been suggested that transcription is regulated by the VEGFR pathway through Ets1 transcription factor, that miR-126 itself negatively regulates the VEGF-R signaling pathway, and that EGFL7 itself regulates Notch signaling. In cancer therapeutic settings, VEGF or VEGF-R directed therapies are thus likely to affect expression of EGFL7 and miR-126. In colorectal cancer (CRC) tissue, miR-126 and EGFL7 are highly expressed in endothelial cells. In studies of XELOX-treated patients, we have previously reported that the miR-126 expression level measured by quantitative in situ hybridization (qISH) is related to poor response (Hansen et al, BMC Cancer 2012, 12:83). Here, we have analyzed miR-126 and EGFL7 expression by qISH and quantitative immunohistochemistry (qIHC), respectively, in 158 patients from two different cohorts with mCRC after treatment with anti-VEGF therapy (Bevacizumab) in combination with chemotherapy. We found that the EGFL7 vessel area (VA) in primary tumors was closely related to treatment response with a median EGFL7 VA in responding patients of 4 (95% CI 4-6) compared to 8.5 (95% CI 7-11) in non-responders, p<0.001. This difference translated into a borderline significant difference in progression-free survival (PFS, p=0.06). Furthermore, a significant relationship between high EGFL7 VA and KRAS mutation was detected (p<0.05). The results showed no significant relationship between the miR-126 VA and the clinical endpoints. Our study suggests a predictive value of EGFL7 in regard to first-line chemotherapy and bevacizumab in patients with mCRC. The strong predictive value of miR-126 found in mCRC patients treated with chemotherapy only was absent, suggesting that the anti-VEGF directed therapy is likely to have affected a key role of miR-126 in regulating the VEGFR pathway.

Citation Format: Boye S. Nielsen, Flemming B. Sorensen, Anders Johnsson, Anders Jakobsen, Torben F. Hansen. MicroRNA-126 (miR-126) and epidermal growth factor-like domain 7 (EGFL7) in Bevacizumab-treated patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-174. doi:10.1158/1538-7445.AM2014-LB-174
Abstract LB-192: Circulating tumor cell clusters are precursors of breast cancer metastasis
Authors: Aceto Nicola , Bardia Aditya , Spencer Joel A. , Wittner Ben S. , Yu Min , Donaldson Maria C. , Pely Adam , Engstrom Amanda , Zhu Huili , Brannigan Brian W. , Kapur Ravi , Stott Shannon L. , Shioda Toshi , Ramaswamy Sridhar , Ting David T. , Lin Charles P. , Toner Mehmet , Haber Daniel A. , Maheswaran Shyamala
Published: May 2015

The metastatic spread of breast cancer, typically to bone, lung, liver and brain, accounts for the vast majority of cancer-related deaths. Breast cancer metastases are thought to be derived primarily from individual migratory cancer cells, reaching distant sites through the bloodstream and initiating proliferation within distant organs. In addition to these single circulating tumor cells (CTCs), CTC-clusters have been detected in the blood of patients with cancer. In patients with breast cancer, we find that presence of such CTC-clusters is correlated with decreased progression-free survival. To study their functional role, we used mouse models, demonstrating that breast cancer cells injected intravascularly as clusters are more prone to survive and colonize the lungs than similarly injected single cancer cells. Primary orthotopic mammary tumors comprised of differentially tagged cells give rise to oligoclonal CTC-clusters, with 40-fold increased metastatic potential to the lung, compared with single CTCs. Using in vivo flow cytometry, we show that CTC-clusters are rapidly cleared from peripheral vessels, consistent with their trapping in small capillaries. Together, our observations suggest that primary tumor cells break off into the vasculature as CTC-clusters, and exhibit greatly enhanced metastatic propensity.

Citation Format: Nicola Aceto, Aditya Bardia, Joel A. Spencer, Ben S. Wittner, Min Yu, Maria C. Donaldson, Adam Pely, Amanda Engstrom, Huili Zhu, Brian W. Brannigan, Ravi Kapur, Shannon L. Stott, Toshi Shioda, Sridhar Ramaswamy, David T. Ting, Charles P. Lin, Mehmet Toner, Daniel A. Haber, Shyamala Maheswaran. Circulating tumor cell clusters are precursors of breast cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-192. doi:10.1158/1538-7445.AM2014-LB-192
Abstract LB-195: Penfluridol, an antipsychotic drug suppresses breast cancer metastasis to brain by inhibiting HER2/Akt/MMP signaling axis in a novel in vivo metastasis model
Authors: Ranjan Alok , Gupta Parul , Srivastava Sanjay K.
Published: May 2015

Breast tumor metastasis is primary cause of cancer-related deaths. Metastasis is a complex process involving cell migration and invasion of tumor cells to distant organs from primary sites. Brain metastasis is a frequent cause of reduced survival of breast cancer patients. In the current study, we evaluated the anti-metastatic effects of penfluridol, a first generation highly potent antipsychotic drug in a novel murine model of breast tumor metastasis to brain. 4T1 breast tumor cells with stable luciferase expression were injected into the left ventricle of mouse heart. The migration of cells to brain was monitored using a non-invasive IVIS bio-luminescent live animal imaging system. To evaluate the anti-metastatic effects, 10mg/kg penfluridol was administered orally to mice every day for 12 days after intra-cardiac injection of tumor cells. The growth rate of metastasized tumors cells in control and treated mice was determined by measuring the luminescence in the brain area in each mouse at different time points. Our results demonstrate that the growth of metastatic brain tumors was reduced by 82% in the mice treated with penfluridol. In addition, treatment of 4T1, MCF-7 or MDA-MB-231 cells with 10µM Penfluridol for 24 hours resulted in the significantly reduced migration and 30-40% reduced invasion of cells, as evaluated by wound healing and Boyden's transwell invasion assay respectively. Penfluridol treatment also reduced the expression of HER2, AKT and MMP-2, 9 in these cells. Interestingly, overexpression of HER2 led to the phosphorylation of Akt, which in turn is known to regulate MMPs. Taken together, our results indicate that the anti-metastatic effects of penfluridol were associated with down regulation of HER2, AKT and MMPs. To the best of our knowledge, our study for the first time demonstrates the anti-metastatic effects of penfluridol in vivo in a novel breast tumor metastasis model. Most importantly, penfluridol is already in clinical use with an established safety record; therefore any positive findings from our studies can be rapidly translated to the clinics for undertaking a clinical trial to treat advanced metastatic breast cancer patients. [Supported in part by R01 grant CA 129038 awarded by National Cancer Institute, NIH]

Citation Format: Alok Ranjan, Parul Gupta, Sanjay K. Srivastava. Penfluridol, an antipsychotic drug suppresses breast cancer metastasis to brain by inhibiting HER2/Akt/MMP signaling axis in a novel in vivo metastasis model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-195. doi:10.1158/1538-7445.AM2014-LB-195