bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3843: Design of a novel three-dimensional biomimetic nano bone model for breast cancer bone metastasis and therapeutics discovery.
Authors: Wang Mian , Fu Sidney W. , Zhang Lijie
Published: August 2014

Breast cancer is the leading cause of cancer-related death in American women, accounting for more than 200,000 new cases and 40,000 deaths annually. Most breast cancer patients will eventually develop complications due to bone metastasis. However, the mechanisms by which breast cancer metastasizes to bone are not well investigated. Traditional tumor metastasis models contain many inherent limitations. Therefore, the objective of this research is to design an innovative biomimetic 3D tissue engineered in vitro bone model via hydroxyapatites (HA), human bone marrow mesenchymal stem cell (hMSC) and natural chitosan hydrogel for breast cancer metastasis study and therapeutics discovery. 3D porous HA-chitosan scaffolds with different crystalline, size and concentration of HA (specifically, 10% and 20% nanocrystalline HA, microcrystalline HA and amorphous HA) were fabricated via a lyophilization procedure. Metastasis breast cancer cells MDA-MB-231 were seeded on all scaffolds and cultured for 4h, 1, 3 and 5 days. The cell adhesion and proliferation results showed that concentration, size and crystallinity of HA have a significant impact on metastasis breast cancer cell behavior. 10% nanocrystalline HA can significantly enhance breast cancer cell attachment when compared to all of other HA. Considering natural bone also consisting of nanocrystalline HA, 10% synthesized nanocrystalline HA in porous chitosan scaffold can create a more biomimetic bone environment for breast cancer bone metastasis study. Furthermore, our bone model was further modified by inducing hMSC osteogenic differentiation in the scaffold resulting in deposition of bioactive protein and calcium by MSC. SEM images of the MSC modified scaffolds revealed that protein and calcium were deposited on both its surface and inner architecture. Two different metastatic breast cancer cells with different metastasis potential (less metastatic MCF-7 and highly metastatic MDA-MB-231) were evaluated in this modified bone model in vitro. The results showed that MDA-MB-231 expanded faster than MCF-7 in the scaffold after 24h culture. Confocal microscopy was used to verify two cells metastasis ability and showed that MDA-MB-231 could migrate deeper in this modified bone model compared to MCF-7. Our results revealed that this model can well reflect the two different metastasis behaviors, making this biomimetic 3D tissue engineered nano bone model promising for future breast cancer cell metastasis study and therapeutics discovery.

Citation Format: Mian Wang, Sidney W. Fu, Lijie Zhang. Design of a novel three-dimensional biomimetic nano bone model for breast cancer bone metastasis and therapeutics discovery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3843. doi:10.1158/1538-7445.AM2013-3843
Abstract 3848: Cryopreserved, viable and architecturally intact lung tissue from patients as a model to study drug therapies.
Authors: Spyropoulos Demetri D. , Denlinger Chadrick E. , Riemer Ellen C. , Newton Danforth A. , Jones E. Ellen , Drake Richard R. , Baatz John E.
Published: August 2014

RATIONALE: Surgery, chemotherapy and radiation have not appreciably improved lung cancer patient survival rates over the past 20 years. Preclinical anti-tumor efficacy testing models could be improved by being rapid, reliable and capable of demonstrating tumor responses to dosing (concentration/timing) and drug interactions, while accounting for inter-patient variation. High-throughput, 3D in vitro systems hold promise as such therapeutic models, but linking these models to clinical responses in patients has been a dilemma.

METHODS: We developed a lung tissue cryopreservation/banking method that optimizes cell viability and maintains tissue architecture after thawing. This method involves the expansion-perfusion of fresh patient lung tissue samples with non-toxic cryoprotectant solutions, followed by controlled-rate freezing of samples.

RESULTS: We used this method to cryopreserve lung tissues from multiple patients. Although inter-patient variability in lung tissue compliance was anticipated, a wide range of expansion volumes can be tolerated without architectural damage. In contrast, tissue integrity was more sensitive to variations in expansion-perfusion rates (as gauged by lymphatic expansion/tearing). Cryosubstitution demonstrated that crystal formation varies with cryoprotectant cocktail and region within the lung, but can be virtually eliminated under certain conditions. From thawed lung tissues, we have 1) isolated and expanded viable lung cell types; 2) isolated decellularized extracellular components; and, 3) reconstituted intact lung tissue with fluorescent-tagged lung tumor cells. We performed patient surgeries/preservations individually, thawing, processing and analysis of lung tissue from multiple patients was performed simultaneously.

CONCLUSION: Cancer cell drug responses are being compared in lung tissues ex vivo versus on standard monolayer culture using advanced proteo-/lipidomic mapping techniques (including MALDI MS and DIGE). Comparing multiple patient-specific lung tissues simultaneously should afford patient population stratification based on molecular profiles and achieve more rapid methods for determining personalized clinical treatment regiments.

Citation Format: Demetri D. Spyropoulos, Chadrick E. Denlinger, Ellen C. Riemer, Danforth A. Newton, E. Ellen Jones, Richard R. Drake, John E. Baatz. Cryopreserved, viable and architecturally intact lung tissue from patients as a model to study drug therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3848. doi:10.1158/1538-7445.AM2013-3848
Abstract 3850: The interaction of RUNX1 with CBFβ-SMMHC during leukemogenesis.
Authors: Zhao Ling , Hyde R Katherine , Alemu Lemlem , Liu P Paul
Published: August 2014

Chromosome 16 inversion is associated with acute myeloid leukemia subtype M4Eo and produces a fusion gene CBFB-MYH11 that contains part of the core binding factor (CBF) β gene CBFB, and part of the smooth muscle myosin heavy chain (SMMHC) gene MYH11. This fusion gene encodes a fusion protein CBFβ-SMMHC, which is oncogenic and binds to the runt domain (RD) of RUNX1, another member of the CBF transcription factor family, resulting in repression of RUNX1 transactivation. We have generated mouse models by conventional and conditional knock-in of the Cbfb-MYH11 fusion gene and demonstrated that Cbfb-MYH11 represses Runx1 function in hematopoiesis and predisposes mice to myeloid leukemia (Castilla et. al., Cell 1996; Nat Genet, 1999).

RUNX1 binding and repression was previously considered a key step in leukemogenesis by CBFβ-SMMHC. In order to dissect the molecular mechanism of RUNX1 and CBFβ-SMMHC interaction during leukemogenesis, we generated a knock-in mouse model with deleted high affinity binding site of Cbfb-MYH11. We found accelerated leukemia development in these mice (Kamikubo et.al., Cancer cell, 2010) suggesting that Cbfb-MYH11 play an independent role apart from Runx1 binding and repression. To test if Runx1 is involved in the leukemia development and progression, we crossed Cbfb-MYH11 knock-in mice with mice harboring one of the two mutant alleles of Runx1 - Runx1+/- and Runx1+/Lzd. Runx1+/- contains a null allele while Runx1+/Lzd contains a knocked-in fusion between the RD of Runx1 and the LacZ gene, which is partially dominant-negative in reporter assays. We have determined the rate and percentage of leukemia development in these mice. We found that the Cbfb-MYH11 mice that were Runx1+/- had a similar rate of leukemogenesis when compared with Cbfb-MYH11 mice that were Runx1+/+. However, the Cbfb-MYH11 mice that were Runx1+/Lzd had significantly delayed leukemogenesis as compared to Cbfb-MYH11 mice that were Runx1+/+. Moreover, some of the Cbfb-MYH11; Runx1+/Lzd mice did not develop leukemia at the end of the one-year observation. We detected a decrease of BrdU incorporation in the bone marrow cells in mice with the Runx1+/Lzd allele, suggesting that the delayed leukemia development resulted, at least in part, from decreased proliferation. These data demonstrated that Runx1 is likely required for leukemogenesis by CBFβ-SMMHC.

Citation Format: Ling Zhao, R Katherine Hyde, Lemlem Alemu, P Paul Liu. The interaction of RUNX1 with CBFβ-SMMHC during leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3850. doi:10.1158/1538-7445.AM2013-3850
Abstract 3858: Chronic myeloid leukemia in the aryl hydrocarbon receptor knockout mouse.
Authors: Butler Ryan , Warner Margaret , Gustafsson Jan-Åke
Published: August 2014

The aryl hydrocarbon receptor (AhR) is a sensor of environmental toxins such as TCDD and its activation by these ligands can lead to immune system impairment, endocrine disruption and cancer. However, the normal physiological functions of AhR are still under investigation. We have previously reported the presence of large uric acid stones in the urinary bladder of AhR-/- mice and have traced this uric acid to rapid degradation of cells. In order to identify the cells that are dying, we examined the immune system of these mice. AhR -/- mice develop splenomegaly, increased number of megakaryocytes, increased circulating white blood cell count, enlarged lymph nodes, abnormalities in the bone marrow with many immature blast cells and severe myeloid cell infiltration into the liver and other tissues. The immune phenotype in these mice worsens as they age. The AhR -/- mouse phenotype, diagnosed as chronic myeloid leukemia, is similar to that of ERβ -/- mice but there is no loss of ERβ in AhR-/- mice. These studies suggest a novel mechanism for the development of chronic myeloid leukemia.

Citation Format: Ryan Butler, Margaret Warner, Jan-Åke Gustafsson. Chronic myeloid leukemia in the aryl hydrocarbon receptor knockout mouse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3858. doi:10.1158/1538-7445.AM2013-3858
Abstract 3860: Furin is required for processing KISS1 to kisspeptins.
Authors: Harihar Sitaram , Pounds Keke M. , Iwakuma Tomoo , Seidah N G. , Welch Danny R.
Published: August 2014

KISS1 is a broadly functional metastatic suppressor that is secreted and processed in the extracellular milieu into small peptides (called kisspeptins (KP)). The enzyme(s) responsible for generation of KP from nascent KISS1 is unknown, although sequence analysis (cleavage at KR or RR dibasic sites) suggests that members of the proprotein convertase family may be involved. We, therefore, hypothesized that enzyme(s) belonging to the proprotein convertase family process KISS1 to generate kisspeptins. To this end, we treated multiple melanomas and breast carcinoma cells overexpressing KISS1 with the proprotein convertase inhibitor Dec-RVKR-CMK and found that KISS1 processing was completely inhibited, strongly consistent with a role for proprotein convertases. To identify specific proprotein convertases responsible for KISS1 processing, we systematically analyzed the mRNA expression of the nine members of this family across numerous cell lines. We found a consistent expression of the three proteases - furin, PC5 and PC7 -suggesting one or combination of these three enzymes to be involved in KISS1 processing. shRNA-mediated knockdown of furin, PC5 and PC7 (individually and in combination) resulted in complete loss of KISS1 processing only in furin knockdown cells; whereas, no effect on processing was observed in PC5 or PC7 knockdown cells. Thus, when combined with previous studies showing post-secretion processing, extracellular furin was identified as the essential enzyme responsible for KISS1 proteolytic cleavage into kisspeptins. [Support: CA134981, Komen SAC110037, National Foundation for Cancer Research, Steiner Family Fellowship in Metastasis Research]

Citation Format: Sitaram Harihar, Keke M. Pounds, Tomoo Iwakuma, N G. Seidah, Danny R. Welch. Furin is required for processing KISS1 to kisspeptins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3860. doi:10.1158/1538-7445.AM2013-3860
Abstract 3864: Blocking hypoxia-induced tumor cell migration by p16/INK4A.
Authors: Lu Yi
Published: August 2014

One general characteristic of fast-growing solid tumors including breast cancer is the development of intratumoral hypoxia. The activation of hypoxia-inducible factor-1α (HIF-1α) by intratumoral hypoxia stimulates a group of downstream genes including vascular endothelial growth factor (VEGF) that are responsible for tumor malignant progression. Loss of p16 expression occurs in many cancers including breast cancer. Our studies showed that, by ectopic expression via adenoviral-mediated gene transfer, p16 downregulates VEGF gene expression by neutralizing the transactivation of HIF-1α, and suppresses breast cancer angiogenesis and metastasis. To investigate whether p16 directly affects one or more aspects of the metastatic cascade, MDA-MB-231 cells infected with lentivirus expressing Tet-on inducible p16 were used to study the p16 effects on growth, adhesion and migration of the breast cancer cells. We found that p16 inhibits MDA-MB-231 cell proliferation (45.6% inhibition) and hypoxia-induced cell migration (36% inhibition), but has no apparent effect on cell adhesion. The p16-mediated inhibition of hypoxia-induced cell migration parallels with p16’s inhibition of HIF-1α transactivation activity. These results indicate that p16’s ability to suppress breast tumor metastasis may result from a combined p16-mediated actions including inhibition of cell proliferation, migration, and angiogenesis. This study suggests that p16 gene therapy may have clinical potentials to treat breast cancer due to p16’s multiple-level anti-cancer capabilities: inhibiting proliferation, inducing apoptosis, blocking tumor angiogenesis, inhibiting cell migration and suppressing metastasis.

Citation Format: Yi Lu. Blocking hypoxia-induced tumor cell migration by p16/INK4A. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3864. doi:10.1158/1538-7445.AM2013-3864
Abstract 3867: Novel mouse models to investigate the molecular pathogenesis of metastatic osteosarcoma.
Authors: Zhao Shuying , Kurenbekova Lyazat , Donehower Lawrence A. , Yustein Jason T.
Published: August 2014

Background: Osteosarcoma (OS) is the primary bone tumor in the pediatric population with the main determinant for patient prognosis being the presence of metastatic disease. Approximately 25-30% of patients will present with metastatic osteosarcoma. Patients with metastatic disease have long-term survival rates often <20%.

Objectives: We hypothesize that the molecular pathogenesis of metastatic osteosarcoma is different from localized disease. We intend this work to provide a relevant, endogenous model of metastatic OS that can be utilized to advance our understanding of the molecular pathogenesis of the disease, insights into novel therapeutic targets and as a pre-clinical model for investigating the efficacy of novel therapies.

Design: We have developed a tissue-specific alteration of the p53 status by using osteoblast specific Cre-recombinase expressing mice to generate progeny that spontaneously form endogenous osteosarcomas. Through the use of a mutated, gain of function form of p53, shown previously to be associated with metastatic disease, we have developed a novel immunocompetent model that significantly enhances the endogenous development of metastatic OS.

Results: Tumor analysis has revealed genetic insights in the metastatic progression of osteosarcoma. These include the significant downregulation of Wnt-signaling inhibitors, such as NKD2, APCDD1 and Wnt5a in the metastatic tumors. Functional studies have determined that overexpression of NKD2, also downregulated in several human OS metastatic cell lines, in metastatic mouse OS cell lines leads to a significant decrease in metastatic lung lesions upon transplantation into immunodeficient mice. Possible NKD2-dependent functions include regulation of not only the Wnt signaling pathway, but also blood vessel formation, regulation of cell migration and cell adhesion.

We have also noted the dysregulation of several critical microRNAs in metastatic OS, including the upregulation of mir-130b in the metastatic lesions. This particular microRNA has recently been shown to have clinical correlation in Ewing's sarcoma, with higher levels of tumor mir-130b having a significantly poorer outcome. We are actively pursuing the role of this (and other) microRNA(s) in the molecular pathogenesis of metastatic osteosarcoma.

Conclusion: This novel model has enabled valuable molecular insights into the development and progression of metastatic OS that can lead to the identification of novel therapeutic targets.

Citation Format: Shuying Zhao, Lyazat Kurenbekova, Lawrence A. Donehower, Jason T. Yustein. Novel mouse models to investigate the molecular pathogenesis of metastatic osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3867. doi:10.1158/1538-7445.AM2013-3867
Abstract 3868: Identification of PHLPP1 as a novel metastasis suppressor in melanoma .
Authors: Yu Yanlin , Lu Andrew , Dai Meng , Merlino Glenn
Published: August 2014

Metastatic dissemination is the cause of nearly all melanoma-related deaths. Despite an improving understanding of genetic lesions present in human melanomas, how these alternations relate to the metastatic process is not well understood. Previously, we found that activation of the PI3K/Akt pathway is a driving force in metastatic melanomas. Here we identify PHLPP1 as a novel metastasis suppressor in melanoma. PHLPP1 was recently identified as a Ser/Thr-specific phosphatase, belonging to pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase family. The two isoforms of this family, PHLPP1 and PHLPP2 control kinase signaling in the AKT family by catalyzing the dephosphorylation of the hydrophobic phosphorylation motif. PHLPP1 can specifically dephosphorylate AKT2 and AKT3, whereas PHLPP2 dephosphorylates AKT1 and AKT3. To study the role of PHLPPs in metastatic melanoma, we first examined the expression of PHLPPs in 30 melanoma metastases, 30 primary melanomas, and 9 normal skin tissues using tissue microarray analysis. We found that PHLPP1 was significantly downregulated or lost in metastatic melanoma samples, but not PHLPP2. Moreover, analysis of various human melanoma cell lines (12 highly metastatic, 14 poorly metastatic melanoma cell lines and 1 normal melanoctye cell line) demonstrated that PHLPP1 expression correlated with metastatic behavior, supporting the notion that PHLPP1 is involved in the metastatic regulation of melanoma. To confirm a functional role for PHLPP1 in metastasis, we forced the expression of PHLPP1 and PHLPP2 in B16 and A375sm melanoma cell lines. Overexpression of PHLPP1 dramatically inhibited metastasis in both cell lines. Unexpectedly, the PHLPP2 isoform had little influence on metastatic ability in both cell lines in vivo, despite the fact that both PHLPP1 and PHLPP2 were able to inhibit in vitro cell proliferation, motility, migration as well as colony formation in soft agar. Notably, introduction of PH mutant forms of PHLPP1 still blunted the metastatic potential of both cell lines, whereas a phosphatase-deficient C-terminal mutant did not affect metastasis. Interestingly, transfected AKT2 active form could rescue PHLPP1-mediaed inhibition of metastasis. Our data demonstrate at the first time that PHLPP1 plays a critical role in inhibiting metastasis through its phosphatase activity, and suggest that PHLPP1 could be a diagnostic marker and therapeutic target for metastatic melanoma.

Citation Format: Yanlin Yu, Andrew Lu, Meng Dai, Glenn Merlino. Identification of PHLPP1 as a novel metastasis suppressor in melanoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3868. doi:10.1158/1538-7445.AM2013-3868
Abstract 3870: Overexpression of SPARC represses MMP-9 mediated metastasis in neuroblastoma.
Authors: Velpula Kiran Kumar , Gorantla Bharathi , Asuthkar Swapna , Bhasin Arnima , Gogineni Venkateswara Rao , Chetty Chandramu , Rao Jasti S.
Published: August 2014

Neuroblastoma (NB) is the most common pediatric solid tumor and arises from defective neural crest cells. NB is characterized by local invasion and metastasis, which is partly regulated by the proteins matrix metalloprotease-9 (MMP-9) and secreted protein acidic and rich in cysteine (SPARC). Although the pleiotropic anti-cancer role of SPARC and its effect on metastasis has been shown in a variety of cancers, the mechanism by which it acts is not very clear. In the present study, we found that the overexpression of SPARC in NB-1649 and SK-AS cell lines downregulated MMP-9 protein and inhibited the expression of other metastasis promoting molecules such as N-cadherin, β-Catenin and FAK. Moreover, SPARC overexpression significantly suppressed MMP-9-mediated migration, invasion and proliferation of NB cells. Interestingly, immunohistochemistry analysis of 36 human NB paraffin-embedded specimens showed that higher expression of MMP-9 was associated with lower levels SPARC. All these data suggest that SPARC and MMP-9 negatively associate to regulate the various stages of tumor progression. Furthermore, computational docking studies showed the existence of structural and physical interaction between SPARC and MMP-9 proteins. This SPARC-MMP-9 interaction was validated by immunoprecipitation and immunohistochemistry analysis in NB-1649 and SK-AS cells. Consistent with the above results, we observed a similar MMP-9-SPARC interaction in intracranial NB tumors of mice and found that the expression of MMP-9 was higher in control mice when compared to SPARC-treated mice. Taken together, our results demonstrate the role of SPARC in negative regulation of MMP-9 and MMP-9-mediated migration, invasion and metastasis of NB cells.

Citation Format: Kiran Kumar Velpula, Bharathi Gorantla, Swapna Asuthkar, Arnima Bhasin, Venkateswara Rao Gogineni, Chandramu Chetty, Jasti S. Rao. Overexpression of SPARC represses MMP-9 mediated metastasis in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3870. doi:10.1158/1538-7445.AM2013-3870
Abstract 3873: MMP9 processing of HSBP1 regulates tumor metastases.
Authors: Choi Seo-Hyun , Lee Hae-June , Jin Yeung Bae , Lee Minyoung , Kim Joon , Yoon Sam S. , Lee Yun-Sil , Lee Yoon-Jin
Published: August 2014

Matrix metalloproteinases regulate pathophysiological events by processing matrix proteins and secreted proteins. Previously, we demonstrated that soluble heat shock protein B1 (HSPB1) is released primarily from endothelial cells (ECs) and regulates angiogenesis via direct interaction with vascular endothelial growth factor (VEGF). Here we report that MMP9 can cleave HSPB1 and release anti-angiogenic fragments, which play a key role in tumor metastasis. We mapped the cleavage sites and explored their physiological relevance during these processing events. HSPB1 cleavage by MMP9 inhibited VEGF-induced ECs activation and the C-terminal HSPB1 fragment exhibited more interaction with VEGF than did full-length HSPB1. HSPB1 cleavage occurs during B16F10 lung metastases in wild-type mice and is decreased in B16F10 lung metastases of MMP9-null mice. Finally, we confirmed that secretion of C-terminal HSPB1 fragment was significantly inhibited lung metastases of B6F10 melanoma cells, compared to full-length HSPB1. These data suggest that in vivo MMP9-mediated processing of HSPB1 acts to maintain an angiogenic balance against tumor metastases. Moreover, these findings potentially explain an anti-target effect for the failure of MMP inhibitors in clinical trials, suggesting that MMP inhibitors may have pro-tumorigenic effects by reducing HSPB1 fragmentation.

Citation Format: Seo-Hyun Choi, Hae-June Lee, Yeung Bae Jin, Minyoung Lee, Joon Kim, Sam S. Yoon, Yun-Sil Lee, Yoon-Jin Lee. MMP9 processing of HSBP1 regulates tumor metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2013-3873
Abstract 3875: Effect of Nutlin and E3 ligase inhibitor on p53 and p21 levels in HDM2 expressing LNCaP cells.
Authors: Shaikh Saamia , Reddy Melanie , Dhandayuthapani Sivanesan , Alagarsamy Karthikeyan , Schmitt Emily , Rathinavelu Appu
Published: August 2014

HDM2 is a human homologue of murine double minute 2 protein (MDM2), which is also a negative regulator for p53 mediated tumor suppressor mechanisms. The latter is involved in regulating cell proliferation and induction of apoptosis as a means of controlling tumor growth. HDM2 can directly bind to p53 and neutralize its ability to induce p21 transcription. HDM2 also contains E3 ligase activity that is responsible for p53 ubiquitination and proteosomal degradation. Hence, compounds that inhibit E3 ligase activity of HDM2 are expected to lead to elevated p53 levels via inhibition of ubiquitination-mediated degradation. Similarly, blocking HDM2 by Nutlin was also expected to restore the levels of p53. According to existing literature, up-regulation of p53 should lead to activation of downstream pathways, such as induction of p21 protein. To confirm this hypothesis the prostate cancer cell line (LNCaP) was treated with HDM2-E3 ligase inhibitor and Nutlin for a 24-hour time period. The levels of p53 and p21 proteins were measured by Western Blot analysis and the mRNA levels were measured by RT-PCR amplification. Our preliminary results showed that HDM2-E3 ligase inhibitor increased p53 levels in LNCaP cells. The increase in p53 levels was greater in Nutlin treated samples compared to E3 ligase inhibited treated samples. In addition, we suspect that induction of p53 expression may also activate downstream pathways that will eventually lead to inhibition of cyclin-dependent kinases (CDKs) and phospho-retinoblastoma (pRB) protein mediated cell proliferation. To confirm our hypothesis further studies will be conducted. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

Citation Format: Saamia Shaikh, Melanie Reddy, Sivanesan Dhandayuthapani, Karthikeyan Alagarsamy, Emily Schmitt, Appu Rathinavelu. Effect of Nutlin and E3 ligase inhibitor on p53 and p21 levels in HDM2 expressing LNCaP cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3875. doi:10.1158/1538-7445.AM2013-3875
Abstract 3876: TRIM16 is a prognostic marker for patients with lymph node metastatic melanoma.
Authors: Cheung Belamy B. , Sutton Selina K. , Tan Owen , Koach Jessica , Liu Bing , Liu Tao , Scolyer Richard A. , Zhang Xu Dong , Marshall Glenn M.
Published: August 2014

The family of tripartite-motif (TRIM) proteins is involved in diverse cellular processes, ranging from innate immunity, oncoprotein, and tumor suppressor roles. TRIM16 overexpression confers retinoid sensitivity in retinoid-resistant cancer cells. Previously, we found a strong correlation between loss of TRIM16 expression and disease progression in two human cancers: neuroblastoma and squamous cell carcinoma of skin. Moreover, TRIM16 acted as a tumor suppressor in both cancers through effects on cell cycle and migration. Here we examined the potential role of TRIM16 as a tumor suppressor in melanoma.

We found that the endogenous TRIM16 expression level in seven melanoma cell lines was significantly lower than in normal human melanocytes. Overexpression of TRIM16 reduced cell viability and proliferation in multiple melanoma tumor cell lines assessed by the Alamar Blue assay, compared to empty vector controls (P < 0.01). Overexpression of TRIM16 also decreases cell migration and induces apoptosis in melanoma cells. Immunohistochemical analysis demonstrated that TRIM16 expression is decreased in human metastatic melanoma tissue samples (Stage 3 and 4) compared to dysplastic compound nevi and primary melanomas (n=91). Most importantly, we have demonstrated that high TRIM16 expression is a favourable prognostic marker of survival (P=0.025) in a cohort of 170 melanoma patients with lymph node metastases. Median survival was 59 months in the high TRIM16 expression group (n=64), compared to 16 months in the low TRIM16 expression group (n=106). We also show overexpression of TRIM16 in melanoma cells lines induced interferon-beta 1 (IFNB1) mRNA expression, and IFNB1 expression is required for inhibition of cell proliferation by TRIM16. IFNB1 expression was also reduced in human metastatic melanoma tissue samples (n=91), and positively correlated with TRIM16 expression in patient samples. Our in vitro and in vivo data suggest that TRIM16 is a prognostic marker for patients with lymph node metastatic melanoma and may play an important role in melanoma tumor metastasis.

Citation Format: Belamy B. Cheung, Selina K. Sutton, Owen Tan, Jessica Koach, Bing Liu, Tao Liu, Richard A. Scolyer, Xu Dong Zhang, Glenn M. Marshall. TRIM16 is a prognostic marker for patients with lymph node metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3876. doi:10.1158/1538-7445.AM2013-3876
Abstract 388: Morphological assessment of tumor-associated vessels: Automated image processing of whole tumor sections in invasive breast cancer.
Authors: Fakhrejahani Elham , Asao Yasufumi , Yamaga Iku , Torii Masae , Takada Masahiro , Kitai Toshiyuki , Sugie Tomoharu , Toi Masakazu
Published: August 2014

Clinical benefit of anti-angiogenic therapies in different cancers is a result of normalizing the tumor vasculature by these agents. However, tumor vascular imaging modalities in human cancers are in their early evaluating phases. Photoacoustic mammography (PAM) is a newly introduced technique which can image the hemoglobin distribution and oxygenation state inside breast tissue with high spatial resolution. We have previously presented the oxygenation data of our first study in breast cancers by PAM in European Congress of Radiology (ECR) 2011. Here we report the morphological assessment of tumor-associated vessels in 81 histological sections of 20 invasive breast carcinomas.

The histological slides were scanned with the magnification of 200 after CD31 immunohistochemical staining. For automated CD31 analysis, 105 jpeg files were extracted from each virtual slide and were changed to binary images with Image Pro-Plus 7.0 software. The optimal hue, saturation and intensity (HSI) of stained structures was selected for each virtual slide. Each binary file was divided into 16 squares of 0.84mm x 0.84 mm size. Total number, area and length of stained structures were calculated in each square.

Patients’ age ranged from 36 to 84 years.19 cases were invasive ductal carcinomas and 1 case was invasive lobular carcinoma. Nine cases were luminal A, 9 cases luminal B and 2 cases were HER-2 subtype. For Ki67 scoring ndex,5 cases had <5%, 9 cases between 5-20% and 6 cases had more than 20%.

There was a wide heterogeneity among total of 2.95 x10 6 analyzed tumor-associated vessels in 81 histological sections.

Table-1 shows the characteristics and distribution pattern. Total number of vessels/mm2 of breast tissue Total area of vessels (μm2)/mm2 of breast tissue Total perimeter of vessels (μm)/mm2 of breast tissue Perimeter/vessel (μm) Area/vessel (μm2) Min 4.6 218.4 65.5 17.8 37.7 Max 87 4369.8 1274.5 45.4 139.3 Average (+SD) 30.7 (+19) 1573.9 (+1060.8) 448.3 (+286.7) 28.9 (+ 4.2) 71.6 (+ 17.9)

Additional analysis of shape and pericyte coverage in these vessels is on its way. In conjunction with oxygenation data obtained by PAM,this can be used as a tumor vessel normalization assessment tool which can be used as a biomarker in anti-angiogenic treatments in breast cancers.

Citation Format: Elham Fakhrejahani, Yasufumi Asao, Iku Yamaga, Masae Torii, Masahiro Takada, Toshiyuki Kitai, Tomoharu Sugie, Masakazu Toi. Morphological assessment of tumor-associated vessels: Automated image processing of whole tumor sections in invasive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 388. doi:10.1158/1538-7445.AM2013-388
Abstract 3880: Exploring the role of up-regulated genes in highly metastatic oral cancer subline using spontaneous metastatic mouse model.
Authors: Muramatsu Tomoki , Kozaki Ken-ichi , Imoto Seiya , Yamaguchi Rui , Miyano Satoru , Inazawa Johji
Published: August 2014

Metastasis is one of the critical factors contributing to poorer prognosis and QOL in cancer patients. Although this mechanism has been gradually revealed, there are few genes found as useful diagnostic markers or molecular targets for clinical application so far. In this study, we attempted to establish in vitro/in vivo metastatic models using a human oral squamous cell carcinoma cell lines (HOC313). We first carried out in vivo selection of high-metastatic tumor cells by means of sequential subcutaneous (s.c.) implantation (1×107 cells) of this cell line into SCID mice, resulting in the establishment of a highly metastatic subline which metastasized to axillary lymph nodes and lung. Moreover, we developed the imaging system using parent cells and highly metastatic cells. Then, to explore metastasis-related genes and signaling pathways, we performed integrative analyses using array CGH, cDNA microarray and the systems biological approach based on their data sets. Then we identified copy number aberrations (CNAs) of chromosome 19 and differentially expressed genes between parent cells and high-metastatic cells. Up regulated genes in high-metastatic cells are related with cell growth, migration, invasion and metastatic ability. Among these, one gene was closely associated with migration, invasion and metastasis. Moreover, when knocking down of this gene, we observed morphological change from mesenchymal phenotype to epithelial phenotype. Further analyses of these genes may ultimately lead to the development of novel modalities of diagnosis and therapy for cancer metastasis.

Citation Format: Tomoki Muramatsu, Ken-ichi Kozaki, Seiya Imoto, Rui Yamaguchi, Satoru Miyano, Johji Inazawa. Exploring the role of up-regulated genes in highly metastatic oral cancer subline using spontaneous metastatic mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3880. doi:10.1158/1538-7445.AM2013-3880
Abstract 3896: A metabolic enzyme as a pro-angiogenic activator in premalignant mammary cells.
Authors: Gonzalez-Guerrico Anatilde M. , Espinoza Ingrid , Lupu Ruth
Published: August 2014

A crucial step in cancer progression is the development of a high-density vascular network, the angiogenic switch. During carcinogenesis breast angiogenesis occurs at hyperplasia before atypia, reaching maximum vascularization between ductal carcinoma in situ and invasive carcinoma. Altered energy metabolism is an early hallmark of several human malignancies. Fatty Acid Synthase (FASN) is a metabolic enzyme that catalyzes the synthesis of Palmitate from Malonyl CoA. With the exception of liver and adipose tissue, normal cells have low levels of FASN and activity. Here we show that FASN expression is progressively up-regulated in the early steps of human breast carcinogenesis. While under hypoxia FASN expression is downregulated in non-transformed breast epithelial cells that form normal ducts. In pre-malignant breast cancer lesions under similar conditions, FASN expression is upregulated. These data suggest a link between FASN-hypoxia-progression. In addition, we observed that in early stages of breast carcinogenesis, FASN regulates the expression of hypoxia genes, which in turn results in increase vascularization and angiogenic markers. Forced expression of FASN in non-transformed breast epithelial cells resulted in increased activity of HIF-1α and upregulation of VEGF expression. Furthermore, FASN-expressing non-transformed breast epithelial cells under hypoxia conditions produced the necessary factors to stimulate angiogenesis, as measured by tube formation and migration in HUVEC cells. These data provide evidence that FASN promotes pro-angiogenesis under hypoxia at the early onset of breast carcinoma; thus, it could be considered as a target for breast cancer chemoprevention.

Citation Format: Anatilde M. Gonzalez-Guerrico, Ingrid Espinoza, Ruth Lupu. A metabolic enzyme as a pro-angiogenic activator in premalignant mammary cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3896. doi:10.1158/1538-7445.AM2013-3896
Abstract 3897: A novel function for threonyl-tRNA synthetase as a stimulator of angiogenesis and endothelial cell migration.
Authors: Williams Tamara , Mirando Adam , Francklyn Christopher , Lounsbury Karen M.
Published: August 2014

In mammalian systems, several aminoacyl-tRNA synthetases are known to have distinct non-canonical functionality. For the first time, we demonstrate that human threonyl-tRNA synthetase (TARS) induces angiogenesis through endothelial cell signaling, and this induction can be blocked by the borrelidin derivative compound BC194. Upon stimulation with vascular endothelial growth factor (VEGF) or tumor necrosis factor-alpha (TNF-α), human endothelial cells secrete TARS into the extracellular space. Extracellular TARS induces and BC194 inhibits angiogenesis in both in vitro tube formation assays and in vivo chick chorioallantoic membrane assays. Furthermore, the anti-angiogenic effects of BC194 are specific to its inhibition of TARS angiogenic activity rather than an indirect stress response. Additional evidence supports a stimulatory role for TARS in endothelial cell migration as a mechanism for its angiogenic activity. Taken together, these findings reveal a previously undefined function for TARS as an angiogenesis inducer stimulated by hypoxic and inflammatory signals. Detecting or intervening in the non-canonical extracellular TARS signaling pathway may thus provide a novel means of diagnosing or treating angiogenesis and inflammatory responses involved in cancer.

Citation Format: Tamara Williams, Adam Mirando, Christopher Francklyn, Karen M. Lounsbury. A novel function for threonyl-tRNA synthetase as a stimulator of angiogenesis and endothelial cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3897. doi:10.1158/1538-7445.AM2013-3897
Abstract 390: Expression of the actin-binding protein Anillin is a prognostic biomarker for primary ER positive breast cancer.
Authors: Magnusson Kristina , Gremel Gabriela , Pontén Fredrik , Jirström Karin
Published: August 2014

Identification of novel prognostic and predictive factors is important for individualizing prognosis and treatment for cancer patients. The present study aimed at examining the potential prognostic value of Anillin (ANLN) expression in breast cancer. ANLN, an actin-binding protein required for cytokinesis, was discovered using the Human Protein Atlas as being differentially expressed in breast cancer and, thus, a potential prognostic biomarker. The purpose of the Human Protein Atlas project (www.proteinatlas.org) is to generate validated antibodies towards the entire human proteome and to use these antibodies for protein profiling in normal tissues and cancer cells. The freely available Human Protein Atlas provides an opportunity to use antibody-based proteomics as a high-throughput strategy to discover novel cancer biomarkers of potential clinical relevance. ANLN has been shown to be up-regulated in several human tumor types including lung, renal and pancreatic tumors. For the purpose of the current study, tissue microarrays with tumor samples from three independent breast cancer cohorts were analyzed by immunohistochemistry (IHC). SiRNA mediated ANLN knockdown of breast cancer cell lines followed by western blot or IHC on cytospin was done for antibody validation. Cell viability assay and cell cycle assay was used to study the function of ANLN knocked breast cancer cells. The majority of breast tumors displayed moderate to strong nuclear positivity in slightly varying fractions, generally below 25%. High nuclear fraction of ANLN was associated with large tumor size, high histological grade, high proliferation and estrogen receptor (ER) negativity. Furthermore, high nuclear expression of ANLN was significantly associated with poor prognosis for patients with ER positive breast tumors in all three cohorts. SiRNA mediated ANLN knockdown was found to induce low cell viability for ER positive breast cancer cells but not for ER negative breast cancer cells. In addition, cell cycle studies showed that ANLN knockdown yielded a shift to G2/M phase, in both ER positive and ER negative breast cancer cells. In conclusion, our results strongly suggest that ANLN can be used as a prognostic biomarker for ER positive breast cancer patients.

Citation Format: Kristina Magnusson, Gabriela Gremel, Fredrik Pontén, Karin Jirström. Expression of the actin-binding protein Anillin is a prognostic biomarker for primary ER positive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 390. doi:10.1158/1538-7445.AM2013-390
Abstract 3905: Radiation-induced angiogenic signaling pathway in endothelial cells isolated from normal and cancer tissue of human breast.
Authors: Oh Eun-Taex , Park Moon-Taek , Song Min-Jeong , Lee Hyemi , Park Heon Joo
Published: August 2014

Despite the strong possibility that the endothelial cells (ECs) of tumors and normal tissues may differ in various aspects, most previous studies on ECs have used normal cells. Here, we purified ECs from tumorous and normal human breast tissues, and studied the effect of radiation on angiogenesis and the relevant molecular mechanisms in these cells. We found that in normal tissue-derived endothelial cells (NECs), 4 Gy irradiation increased tube formation, MMP-2 expression and ERK pathway activation. In cancer-derived endothelial cells (CECs), however, 4 Gy irradiation significantly reduced tube formation, increased the production of angiostatin and interleukin-6 (IL-6), and up-regulated AKT and JNK pathway activation. Knockdown experiments showed that siMMP-2 efficiently inhibited tube formation by irradiated NECs, while siPlasminogen effectively attenuated the irradiation-induced suppression of tube formation and the up-regulation of angiostatin in CECs. Moreover, siIL-6 clearly inhibited the irradiation-induced generation of angiostatin in CECs. Inhibition of ERK with a pharmacological inhibitor or siRNAs markedly suppressed irradiation-induced tube formation and MMP-2 up-regulation in NECs, while the inhibition of either AKT or JNK with pharmacological inhibitor or siRNA treatment of CECs clearly attenuated the irradiation-induced inhibition of tube formation and the up-regulation of angiostatin and IL-6. These observations collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy.

Citation Format: Eun-Taex Oh, Moon-Taek Park, Min-Jeong Song, Hyemi Lee, Heon Joo Park. Radiation-induced angiogenic signaling pathway in endothelial cells isolated from normal and cancer tissue of human breast. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3905. doi:10.1158/1538-7445.AM2013-3905
Abstract 3918: Validation of ATX-Red imaging as a potential companion diagnostic for autotaxin-expressing cancers.
Authors: Madan Damian , Ferguson Colin , Lee Won Yong , Testa Charles , Prestwich Glenn
Published: August 2014

Autotaxin (ATX), an autocrine motility factor highly upregulated in metastatic cancer, is a lysophospholipase D that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (ATX-Red) that enables visualization of ATX activity in vivo. ATX-Red exhibits minimal fluorescence until it is activated by ATX, which significantly increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with ATX-expressing breast cancer orthotopic tumors, ATX activated ATX-Red fluorescence. Injection of ATX-Red into the tail vein of tumor-bearing mice resulted in high fluorescence in the tumor and low fluorescence in healthy tissues. Indeed, tumor fluorescence directly correlated with ATX activity and expression levels. Pretreatment of mice with a known ATX inhibitor markedly and selectively decreased tumor fluorescence. Taken together, the data show that ATX-Red is an effective, non-invasive, and selective tool that enables intravital visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo in real time. ATX-Red has the potential to (1) identify pathological levels of ATX in specific tissues, (2) monitor the time course of therapeutic efficacy of candidate ATX inhibitors in living animal models of human disease, (3) serve as a companion diagnostic for following treatment regimens that target ATX in human patients and (4) serve as a companion diagnostic to select patients that would benefit from ATX-directed therapy.

Citation Format: Damian Madan, Colin Ferguson, Won Yong Lee, Charles Testa, Glenn Prestwich. Validation of ATX-Red imaging as a potential companion diagnostic for autotaxin-expressing cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3918. doi:10.1158/1538-7445.AM2013-3918