bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4998: NFAT signaling modulates tumor microenvironment.
Authors: Kaunisto Aura , Toker Alex
Published: August 2014

Nuclear factor of activated T cells (NFAT) transcription factors regulate several cancer-related processes, such as migration and invasion. We have shown that NFAT is both necessary and sufficient to drive invasive migration of breast carcinoma cells. NFAT target genes that contribute to this phenotype encode cyclo-oxygenase-2, the enzyme required for the production of prostaglandin E2, and autotaxin, which catalyzes the synthesis of lysophosphatidic acid. NFAT also promotes tumor progression downstream of vascular endothelial growth factors and contributes to angiogenesis and lymphangiogenesis, though the underlying molecular mechanisms that control these phenotypes have remained unknown. In our present studies, we have identified novel NFAT target genes whose products are likely to modify tumor microenvironment through paracrine signaling to immune cells. We have generated stable pools of breast cancer cells with inducible lentiviral silencing or overexpression of NFAT and performed quantitative real-time RT-PCR to identify NFAT-responsive genes that code for soluble factors. This approach yielded several novel candidates, including those encoding cytokines and chemokines. The contribution of these NFAT-regulated gene products to tumor-stromal interactions is currently under investigation. Our studies propose a central role for NFAT in modulating tumor microenvironment.

Citation Format: Aura Kaunisto, Alex Toker. NFAT signaling modulates tumor microenvironment. [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 4998. doi:10.1158/1538-7445.AM2013-4998
Abstract 500: Role of R-spondin4 in Wnt/β-catenin signaling and cell proliferation in breast cancer.
Authors: Lu Wenyan , Lin Cuihong , King Taj K. , Li Yonghe
Published: August 2014

R-spondin 4 (Rspo4) is a member of the Rspo protein family, which is another group of secreted proteins that can activate Wnt/β-catenin signaling. Although it is well recognized that Wnt/β-catenin signaling is important for breast cancer development and progression, the role of Rspo4 in breast cancer is unclear. In the present study, we found that Rspo4 was expressed in breast cancer cell lines, but absent in normal mammary epithelial cells (HMECs). We also found that Rspo4 expression was up-regulated in human malignant breast tissues. Moreover, Rspo4 acted synergistically with Wnt3A to activate Wnt/β-catenin signaling in breast cancer cells. Conversely, reducing endogenous Rspo4 with siRNAs in human breast cancer cell lines suppressed Wnt/β-catenin signaling and cell proliferation. These data suggest that Rspo4 functions as a positive regulator of breast cancer proliferation by activating Wnt/β-catenin signaling.

Citation Format: Wenyan Lu, Cuihong Lin, Taj K. King, Yonghe Li. Role of R-spondin4 in Wnt/β-catenin signaling and cell proliferation in 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 500. doi:10.1158/1538-7445.AM2013-500
Abstract 5002: Intermittent hypoxia selects for increased carcinogenicity in breast epithelial cells.
Authors: Verduzco Daniel , Gatenby Robert , Gillies Robert
Published: August 2014

The hypoxic regions of solid tumors are hotbeds of drug resistance and selection. Tumors with highly hypoxic regions correlate with poor prognosis and increased aggressiveness. The exact molecular mechanisms of selection have not been elucidated. Recent studies have demonstrated that reoxygenation causes widespread DNA damage. Additionally, hypoxia suppresses the DNA damage machinery. Because cells within a solid tumor do not receive a consistent amount of oxygen, cycles of acute hypoxia and reoxygenation occur, a phenomenon termed intermittent hypoxia (IH). We predict that this may lead to widespread unrepaired DNA damage that consistently selects for populations adapted to survive within harsh tumor microenvironmental conditions.

We tested three breast cell lines - one aggressive carcinoma (MB 231), one DCIS line, and one non-transformed epithelial cell line (MCF10A) - for DNA damage and transformation after multiple cycles of IH. Cells were incubated under 0.2% hypoxia for 16 hours and reoxygenated for 8, for multiple cycles. Multiple clones of each cell line selected under IH conditions were then isolated. MCF10A IH-selected clones exhibit increased resistance to etoposide relative to unselected clones. Two of these clones were selected for additional phenotyping, and exhibited constituitive carbonic anhydrase 9 expression, as measured by qRT-PCR. Resistance to methotrexate, docetaxel, and hypoxia induced cell death was also observed, but growth-factor independent proliferation was not detected. Additionally, genomic analyses have revealed loss of the tp53 and e-cadherin loci after intermittent hypoxia. Consistent with this, we detect loss of p53 and e-cadherin mRNA levels and anchorage independent growth. These data increase our understanding of why hypoxia creates more aggressive cancers and poorer prognoses, and how de novo drug resistance arises.

Citation Format: Daniel Verduzco, Robert Gatenby, Robert Gillies. Intermittent hypoxia selects for increased carcinogenicity in breast epithelial 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 5002. doi:10.1158/1538-7445.AM2013-5002
Abstract 5021: Temozolomide-induced conversion of glioma cells into glioma “stem-like” cells with enhanced invasion properties.
Authors: Ahmed Atique U. , Auffinger Brenda , Tobias Alex , Thaci Bart , Han Yu , Lesniak Maciej S.
Published: August 2014

Glioblastoma multiforme patients present a poor prognosis due to therapeutic resistance and tumor recurrence after surgical resection. It has been suggested that gliomas are driven by a rare subset of tumor cells, known as cancer stem cells (CSCs). These tumor-initiating cells are considered to be responsible for sustaining long-term tumor growth and disease recurrence. Today, two popular hypotheses try to explain the origin of CSCs: one postulates that CSCs represent an adult stem cell population with tumorigenic alterations, while the second argues that CSCs were once differentiated tumor cells that have re-initiated a self-renewal program. In this study, we first show that exposure to the therapeutic dose of 5 μM (the peak concentration in patient's cerebral spinal fluid) or 50 μM (the concentration in patient's blood) of temozolamide (TMZ), the most commonly used alkylating agent to treat GBM patients, consistently increase the CSC pool over time both in vitro and in vivo. Second, lineage-tracing analysis of genetically tagged glioma cells suggests that TMZ induces a phenotypic shift in the non-CSC population to mimic that of CSCs. These newly formed CSC-like cells co-express markers of pluripotency and stemness (CD133, CD15, Sox-2, Oct-4 and Nestin), a classical characteristic of CSCs. We further show that intracranial implantation of TMZ-treated non-CSC cells in athymic nude mice result in more efficient grafting (100% grafting as compared to 60% for DMSO treated control group). Moreover, these TMZ-treated non-CSC xenografts demonstrate extremely invasive characteristics, which is similar to the CSC enriched xenografts. In addition, they express elevated levels of HIF2α, a critical factor for maintaining the CSC pool. These findings strongly corroborate that chemotherapy-induced selection pressure can facilitate a CSC specific ‘niche’, which may provide the necessary signals to induce an interconversion between differentiated tumor cells and cancer “stem like” cells, leading to a more infiltrative glioma phenotype thereby resulting in therapeutic relapse and may frustrate our attempts to develop effective anti-glioma therapy.

Citation Format: Atique U. Ahmed, Brenda Auffinger, Alex Tobias, Bart Thaci, Yu Han, Maciej S. Lesniak. Temozolomide-induced conversion of glioma cells into glioma “stem-like” cells with enhanced invasion properties. [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 5021. doi:10.1158/1538-7445.AM2013-5021
Abstract 5033: Modeling Ewing's sarcoma and tolerance of EWS-FLI1 with neural crest stem cells.
Authors: Mosher Jack T. , Chen Victor S. , Lawlor Elizabeth R.
Published: August 2014

Ewing's sarcoma (ES) is a highly malignant bone and soft tissue tumor that primarily affects children and young adults. These tumors are characterized by chromosomal translocations, most commonly t(11;22)(q24;q12), which generates the EWS-FLI1 fusion oncogene. Despite its oncogenic potential, the fusion protein induces cell cycle arrest or apoptosis in most non-transformed cells unless mutations in tumor suppressors such as p16 are also present. However, only a minority of patients (∼25%) have such mutations at diagnosis, suggesting other mechanisms for cellular tolerance must be present in the cell of origin. Currently, the cell of origin of these tumors is controversial, with data supporting both a mesenchymal and/or neural crest origin. To assess the potential to model ES using mouse neural crest stem cells (NCSCs) and elucidate the mechanism(s) of EWS-FLI1 tolerance, primary fetal and adult mouse NCSCs were transduced with a V5-tagged EWS-FLI1 2a eGFP construct. Both wild-type fetal and postnatal NCSCs tolerated EWS-FLI1 expression, with 40-60% of the stem cell-derived neurospheres positive for GFP. There was no difference in neurosphere forming ability between cells infected with EWS-FLI1 or an empty vector, suggesting EWS-FLI1 expression was not toxic to these cells. EWS-FLI1 expression was maintained as the stem cells self-renewed, with GFP-positive primary neurospheres giving rise to GFP-positive secondary and tertiary neurospheres but there was no difference in the frequency of GFP-positive and -negative secondary neurospheres. EWS-FLI1 expression was confirmed by quantitative PCR and the V5 tag was localized to the nucleus by immunocytochemistry, consistent with transcription factor function. EWS-FLI1-mediated gene expression analysis is currently ongoing. Interestingly, the tolerance of EWS-FLI1 was dramatically reduced in Bmi-1 deficient NCSCs. Bmi-1 is known to function cooperatively with multiple other oncogenes to mediate transformation but its role in tolerance has not been studied. In the absence of Bmi-1 only 8% of the fetal neurospheres were positive for GFP, a large decrease compared to 40% of the wild-type neurospheres. Taken together, these data are consistent with a neural crest origin for ES and demonstrate a critical role for Bmi-1-mediated tolerance to EWS-FLI1. Work is ongoing to determine whether EWS-FLI1 can transform these cells and to assess the full utility of this novel system to model ES and genetically assess important regulators of EWS-FLI1 function.

Citation Format: Jack T. Mosher, Victor S. Chen, Elizabeth R. Lawlor. Modeling Ewing's sarcoma and tolerance of EWS-FLI1 with neural crest stem 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 5033. doi:10.1158/1538-7445.AM2013-5033
Abstract 5038: Probing network fragilities in neuroblastoma by synergistic drug combinations.
Authors: Radic Branka , Rix Uwe , Kubicek Stefan , Superti-Furga Giulio
Published: August 2014

Neuroblastoma is the most common tumor in infants younger than 1 year of age and one of the most common solid tumors in childhood. The disease is highly variable: while spontaneous tumor regression occurs more often than in any other cancer type, patients with more severe variant face therapy-resistant recurrence. Therefore we have set out to develop new therapeutic approaches to the disease with a particular focus on aggressive subtypes. Using a high-throughput systems level approach we have identified a novel synergistic combination of approved drugs and compounds in clinical trials.

Our initial screen assessed the potency of a panel of clinically applicable drugs by proliferation assays versus a defined panel of isogenic cell lines that are representative of the different neuroblastoma variants. We identified potential drugs combinations by three-dimensional dose response matrices and adopted Bliss values as a quantitative measure of the synergy. The striking synergy was observed in SH-SY5Y cell line using combinations of EGFR/HER2 inhibitors with the apoptosis inducing agents. We were able to validate the observation using colony formation assay. In contrast to single drug treatment, the combination completely abolished the clonogenic growth of neuroblastoma cells, showing the most tangible evidence of the synergy.

In order to evaluate our results in more aggressive disease subtypes, we tested our combination in SH-SY5Y cell line with transiently overexpressed N-Myc and in N-Myc-amplified IMR5-75 cell line. Notably, even in this setting we could recapitulate our findings, suggesting that this combination of drugs is highly potent in the chemo-resistant tumor subtypes as well. The same effect was observed when we transiently overexpressed TrkA in SH-SY5Y, mimicking yet another neuroblastoma variant.

Pursuing the exact mechanism of the synergy, we have tested the combination in breast cancer cells. To our surprise, the synergistic effect was still present in HER2 negative cell line, but completely absent in the HER2 positive one. We also exchanged the dual EGFR/HER2 inhibitor drug with more potent EGFR inhibitors to conclude that a combination seems to be specific to a particular set of compounds and not necessarily to their main target spectra.

Our synergistic combination is highly potent in a large number of neuroblastoma conditions. Strong synergistic effect can be reached at doses much lower than required for the single drug treatment. Animal models that we are currently testing will enable quick translation of our data in clinics.

Citation Format: Branka Radic, Uwe Rix, Stefan Kubicek, Giulio Superti-Furga. Probing network fragilities in neuroblastoma by synergistic drug combinations. [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 5038. doi:10.1158/1538-7445.AM2013-5038
Abstract 5047: Impact of kidney progenitor cell differentiation status in Wilms tumorigenesis.
Authors: Huang Le , Mokkapati Sharada , Hu Qianghua , Huff Vicki
Published: August 2014

Wilms Tumor (WT) is a pediatric cancer of the kidney and one of the most common solid tumors of childhood. It is thought to arise from the undifferentiated renal mesenchyme, however, it is unknown which mesenchymal cell type can give rise to WT. The recent identification of different subsets of human WTs with defined gene expression signature, histology and clinical outcomes suggests that the cell differentiation status may be an important factor in the etiology of these subsets. Therefore, we hypothesize that WT can arise from fetal kidney cells at different differentiation stages and that different progenitor populations will result in different types of tumors in biology, pathology and molecular signature.

Our lab has generated the first genetic WT mouse model via somatic and mosaic ablation of Wilms Tumor 1 (Wt1), using a Wt1 conditional knockout allele with a ubiquitous tamoxifen-inducible CGH-Cre, and Igf2 biallelic expression. In this model, all types of embryonic kidney cells were randomly targeted. With this successful approach, we now have the ability to test the effect of introducing these alterations (Wt1 ablation in the context of Igf2 up-regulation or activation of β-Catenin) into kidney nephrogenic (Cited1-CreERT2) or stromagenic (Foxd1-CreERT2) progenitor cells. Very interestingly, when the mutations were introduced into the nephrogenic progenitors (Cited1-CreERT), WTs develop. These tumors express human WT markers and show a tubular histology with predominant epithelial cells, which histologically corresponds to the epithelial subset of human WTs. To further characterize the biology of these tumors, we will perform gene expression analysis and proteomic microarray analysis to identify aberrantly dysregulated signaling pathways.

This study will provide insight into the cellular etiology of WT which may help to identify new therapeutic approaches.

Citation Format: Le Huang, Sharada Mokkapati, Qianghua Hu, Vicki Huff. Impact of kidney progenitor cell differentiation status in Wilms tumorigenesis. [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 5047. doi:10.1158/1538-7445.AM2013-5047
Abstract 5048: mTORC1/mTORC2 signaling in pediatric low grade glioma and pilocytic astrocytoma.
Authors: Shah Smit , Hutt Marianne , Karajannis Matthias A. , Raabe Eric , Eberhart Charles G. , Rodriguez Fausto J.
Published: August 2014

Pilocytic astrocytoma is the most frequent low grade glioma in children and young adults. Previous studies support a role for MAPK pathway signaling, and more recently AKT/mTOR, in its biology. mTOR operates as part of two different multiprotein signaling complexes, mTORC1 and mTORC2. In the current study we evaluated mTORC1/mTORC2 pathway activity in pediatric low grade astrocytoma. We used formalin-fixed paraffin embedded human pilocytic astrocytomas for immunohistochemical studies in tissue microarrays. Moderate (2+) to strong (3+) immunostaining was observed for pS6 in 66/114(58%), p4EBP1 in 35/116(30%), pElF4G in 66/113 (58%), and mTOR (total) in 52/114 (46%), supporting mTOR pathway activation in a subset of pediatric astrocytomas. Specifically, there was increased immunoreactivity in pilocytic astrocytomas of the optic nerve proper (pS6 39/54(72%), p4EBP1 22/54 (41%), 4IF4G 41/53 (77%), and mTOR 28/54 (52%)), a difference that was statistically significant for pS6, p4E-BP1 and pEIF4G (p<0.05, Chi Square). Next, we studied total levels of Raptor (mTORC1 component) and Rictor (mTORC2 component). We observed moderate to strong Raptor reactivity in 81/142 (57%) and Rictor in 69/141(49%) pilocytic astrocytomas. By contrast, we observed increased Rictor immunostaining in 90/144 (63%) diffuse gliomas grade II-IV and Raptor in 28/147 (19%), a difference that was statistically significant between the two groups (p<0.001, Chi Square). Next we studied in vitro the effect of mTORC1 blockade in pediatric low grade glioma cell lines Res 186 and Res 259 with a TORC1 inhibitor. Using the MTT assay, Res 186 cell growth was inhibited with 1nM and 10nM drug concentrations, compared with DMSO (p<0.005, t-test), but the effect was less pronounced in Res 259. Additionally, pS6 levels were decreased by Western Blot in Res 186 cells, but not in Res 259, consistent with mTORC1 inhibition of Res 186 cells. The findings suggest that the mTOR pathway is active in a subset of pediatric low grade gliomas, that it varies by clinicopathologic subtype, and that it is primarily mediated by mTORC1 compared with other glioma subtypes. Furthermore, mTOR may represent a therapeutic target in pediatric low grade glioma that merits further investigation.

Citation Format: Smit Shah, Marianne Hutt, Matthias A. Karajannis, Eric Raabe, Charles G. Eberhart, Fausto J. Rodriguez. mTORC1/mTORC2 signaling in pediatric low grade glioma and pilocytic astrocytoma. [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 5048. doi:10.1158/1538-7445.AM2013-5048
Abstract 5052: Maximal SHH-driven medulloblastoma growth requires coordinate CXCR4 activation.
Authors: Ward Stacey , Sengupta Rajarshi , Rubin Josh
Published: August 2014

Medulloblastoma is the most common malignant brain tumor of childhood. Even with current intensive treatments there is still significant morbidity and mortality associated with this diagnosis. Recent hope for improved outcome derives from the stratification of medulloblastoma into four molecular subtypes and the promise that subtype-specific targeted therapy will be more efficacious and less toxic. However, even within specific subtypes there can be variable outcomes suggesting that further subclasification will be required. We have shown that CXCR4, a G-protein coupled receptor important for neuronal cell migration, is differentially expressed within the Sonic hedgehog (SHH) subtype of medulloblastoma (CXCR4hi and CXCR4lo) and that activation of CXCR4 was required for maximal medulloblastoma growth in vitro and in vivo. Here we investigate the molecular basis for the interaction between the SHH and CXCR4 pathways and the therapeutic potential of combined SHH and CXCR4 antagonism for the treatment of medulloblastoma.

Using primary cultures of SHH-driven medulloblastoma cells as well as their normal developmental counterpart, cerebellar granule precursor cells (GPCs), we found that SHH activation enhances CXCR4 surface localization and promotes prolonged CXCL12-induced GαI signaling. We hypothesized that this was due to SHH inhbition of CXCR4 desensitization and investigated the effects of SHH treatment on proteins involved in CXCR4 trafficking including: ferritin heavy chain, GRKs, arrestins and CXCR7. We found that while SHH had no effect on ferritin heavy chain expression, It did alter the abundance of a subset of GRKs, arrestins and CXCR7. In light of these data we developed a model for multi-tiered regulation of CXCR4 signaling by the SHH pathway that suggested combined SHH and CXCR4 antagonism would have a superior anti-tumor effect compared to SHH antagonism alone. We verified this by in vivo treatment of primary murine SHH-driven (SmoA1) medulloblastoma xenografts with the SHH and CXCR4 antagonists vismodegib and plexiflor. Together these data identify new targets and a new approach for the treatment of SHH-driven medulloblastoma.

Citation Format: Stacey Ward, Rajarshi Sengupta, Josh Rubin. Maximal SHH-driven medulloblastoma growth requires coordinate CXCR4 activation. [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 5052. doi:10.1158/1538-7445.AM2013-5052
Abstract 5072: Human tumor derived Oncoprint® platform predicts molecular mechanism of sensitivity and resistance to Fragmin in pancreatic cancer.
Authors: Sundaram Mallikarjun , Thiyagarajan Saravanan , Dhandapani Muthu , Brijwani Nilesh , Prasath Arun , Thayakumar Allen , Kamal Ameer , Radhakrishnan Padhma , Majumder Biswanath , Ulaganathan Baraneedharan , Majumder Pradip K.
Published: August 2014

One of the formidable challenges in cancer therapeutics is developing and validating a highly effective preclinical testing platform. However, existing in vitro and in vivo testing platforms show limited success. In this context, we have developed Oncoprint®, a novel clinically relevant preclinical platform demonstrated to have very high correlation to clinical outcome for chemotherapeutics as well as targeted drugs in multiple solid and hematological cancers (with overall sensitivity of 88% and specificity of 90% in more than 500 patients). Oncoprint® is a rapid reproducible ex-vivo tumor explant system, developed to mimic native tumor microenvironment. By including autologous paracrine growth factors, cancer specific customized matrix support, autologous immune environment along with other growth promoting conditions, significant improvements were observed in viability, proliferation of tumors including retention of tumor/stroma, cancer phenotypes, integrity at micro-architecture level and maintenance of functional signaling network. Using this platform we assessed the anti-tumor efficacy of Fragmin (a low molecular weight heparin-LMWH) alone as well as in combination with various Standard-Of-Care drugs (SOCs) and compared the outcome with efficacy derived from the use of SOCs alone in twenty patient derived pancreatic tumors. Clinical trial data indicates that Fragmin when administered in patients with advanced pancreatic cancer decreased levels of circulating-tissue factor antigen and attenuated induction of cellular invasion in their blood.

Oncoprint data indicates that Fragmin has potent anti-angiogenic activity as a single agent (in ∼ 77% tumor samples) and enhanced anti-proliferative and apoptotic effect in combination with SOC (Cisplatin and Gemcitabine) compared to SOC alone in ∼ 30% of these pancreatic tumors. . Our study has been powered to include 75-100 patient derived refractory pancreatic tumors for further evaluation. Detailed molecular profiling studies are underway to understand the mechanism of action of this drug to delineate the biology behind response in the responder sub-population.

Citation Format: Mallikarjun Sundaram, Saravanan Thiyagarajan, Muthu Dhandapani, Nilesh Brijwani, Arun Prasath, Allen Thayakumar, Ameer Kamal, Padhma Radhakrishnan, Biswanath Majumder, Baraneedharan Ulaganathan, Pradip K. Majumder. Human tumor derived Oncoprint® platform predicts molecular mechanism of sensitivity and resistance to Fragmin in pancreatic 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 5072. doi:10.1158/1538-7445.AM2013-5072
Abstract 509: RNF115/BCA2 E3 ubiquitin ligase promotes breast cancer cell proliferation through targeting p21Waf1/Cip1 for ubiquitin-mediated degradation .
Authors: Wang Zehua , Seth Arun , Chen Ceshi
Published: August 2014

The E3 ubiquitin ligase Ring Finger Protein 115 (RNF115), also known as Breast Cancer Associated gene 2 (BCA2), has previously been reported to be overexpressed in estrogen receptor (ERα) positive breast tumors and to promote breast cell proliferation; however, the mechanism is unknown. In this study, we demonstrated that silencing of BCA2 by siRNA in two ERα-positive breast cancer cell lines MCF7 and T47D decreases cell proliferation and increases the protein levels of the cyclin-dependent kinases inhibitor p21Waf/Cip1. The protein stability of p21 was negatively regulated by BCA2. BCA2 directly interacts with p21 and promotes p21 ubiquitination and proteasomal degradation. Knockdown of p21 partially rescues the cell growth arrest induced by the BCA2 siRNA. These results suggest that BCA2 promotes ERα-positive breast cancer cell proliferation at least partially through down-regulating the expression of p21.

This study was supported by National Nature Science Foundation of China (81072162, 81120108019, and U1132605) .

Citation Format: Zehua Wang, Arun Seth, Ceshi Chen. RNF115/BCA2 E3 ubiquitin ligase promotes breast cancer cell proliferation through targeting p21Waf1/Cip1 for ubiquitin-mediated degradation . [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 509. doi:10.1158/1538-7445.AM2013-509
Abstract 5091: Potent anti-tumor activity of blocking stromal Dll4 in ovarian xenograft models.
Authors: Kuhnert Frank , Chen Guoying , Thurston Gavin
Published: August 2014

Potent anti-tumor activity of blocking stromal Dll4 in ovarian xenograft models

Delta-like ligand 4 (Dll4) is an emerging anticancer target given its predominant tumor vasculature expression and its role in regulating angiogenic sprouting. We have previously demonstrated that pharmacological blockade of the Dll4-Notch axis results in an excessive production of aberrant non-functional tumor vessels, and these changes were associated with reduced tumor growth. Using VelocImmune® mice, we identified a fully human IgG1 monoclonal antibody, termed REGN421/SAR153192, which binds human Dll4 and potently neutralizes Notch signaling. REGN421 treatment caused potent and dose-dependent inhibition of a number of human tumor xenografts grown in immunodeficient mice engineered to express human Dll4. In the current study, we found that REGN421 treatment of ovarian xenograft models produces potent anti-tumor effects that are dependent on targeting Dll4 in the tumor stroma as opposed to tumor cell-expressed Dll4. In particular, REGN421 treatment (2.5 mg/kg, once weekly) of humanized Dll4 mice bearing established subcutaneous TOV-112D or intraperitoneal A2780 human tumor xenografts resulted in growth inhibition of 86% and 83%, respectively. The inhibition of ovarian tumor growth by REGN421 was associated with a marked increase in tumor vascular structures but reduced vascular perfusion, consistent with the function of Dll4 as a regulator of angiogenic sprouting. Similar anti-tumor effects were observed by strictly targeting stromal Dll4 with a mouse Dll4-specific surrogate antibody REGN1035 in SCID mice bearing OVCAR3 tumors. In contrast, the specific blockade of tumor cell-expressed human Dll4 did not exhibit any appreciable anti-tumor activity, indicating the lack of tumor growth-promoting autocrine Dll4-Notch tumor cell signaling in these models. Finally, the combined treatment of Dll4 antibody with the anti-VEGF agent ziv-aflibercept (VEGF Trap) resulted in enhanced anti-tumor effects and virtually the complete suppression of intraperitoneal A2780 tumor growth, suggesting clinical benefit for the combined blockade of VEGF and Dll4 in ovarian cancer. These studies lend further support for the therapeutic targeting of Dll4 as a promising new angiogenesis-based anticancer strategy in ovarian cancer, both as a single agent and in combination with anti-VEGF agents.

Citation Format: Frank Kuhnert, Guoying Chen, Gavin Thurston. Potent anti-tumor activity of blocking stromal Dll4 in ovarian xenograft models. [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 5091. doi:10.1158/1538-7445.AM2013-5091
Abstract 5099: Direct renin inhibitor attenuates rat hepatocarcinogenesis via anti-angiogenic activity.
Authors: Aihara Yosuke , Yoshiji Hitoshi , Noguchi Ryuichi , Kaji Kosuke , Namisaki Tadashi , Moriya Kei , Kitade Mitsuteru , Fukui Hiroshi
Published: August 2014

Direct renin inhibitor (DRI), aliskiren, is widely used as an anti-hypertensive agent in the clinical practice, and several reports have shown that renin plays an important role in several pathological processes. However, the role of renin in the hepatocarcinogenesis is still obscure. The aim of the current study was to examine the effect of DRI on the hepatocarcinogenesis in the rat model. Hepatocarcinogenesis model was induced in male F344 rats by the choline-deficient L-amino acid-defined diet for 12 weeks. The effect of DRI at clinically comparable low dose on the hepatocarcinogenesis was examined especially in conjunction with neovascularization. DRI exerted a marked inhibitory effect on the glutathione-S-transferase placental form (GST-P)-positive preneoplastic lesions along with suppression of the neovascularization in a dose-dependent manner. DRI also inhibited the hepatic expressions of angiotensin-II (AT-II), and vascular endothelial growth factor (VEGF). Our in vitro study showed that renin significantly augmented the in-vitro angiogenesis, whereas, interestingly, DRI did not inhibit the renin-induced the endothelial cell proliferation even at high dose. These results indicate that renin plays a pivotal role in hepatocarcinogenesis at least partly mediated by pro-angiogenic activity. Since DRI is already widely used in the clinical practice with safety, this drug may represent a potential new strategy against the hepatocarcinogenesis in the future.

Citation Format: Yosuke Aihara, Hitoshi Yoshiji, Ryuichi Noguchi, Kosuke Kaji, Tadashi Namisaki, Kei Moriya, Mitsuteru Kitade, Hiroshi Fukui. Direct renin inhibitor attenuates rat hepatocarcinogenesis via anti-angiogenic activity. [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 5099. doi:10.1158/1538-7445.AM2013-5099
Abstract 5100: MMP-2 suppression inhibits α5β1 integrin and IL-3-mediated Stat5 activation and proliferation of endothelial cells.
Authors: Maddirela Dilip Rajasekhar , Kesanakurti Divya , Chetty Chandramu , Rao Jasti S.
Published: August 2014

Endothelial cell (EC) proliferation is a major component of angiogenesis in glioblastoma multiforme (GBM) and is regulated by several growth factors including IL-3. In this study, we have evaluated the effect of conditioned media (CM) obtained from MMP-2 knockdown xenograft 4910 and 5310 cell lines by pMMP-2 siRNA (pM.si) on the growth of ECs. Western blot analysis of CM from pM.si-treated 4910 and 5310 cells showed a significant downregulation of MMP-2 and IL-3 expression compared to mock- and pSV (scrambled vector)-CM. Proliferation decreased in ECs cultured on pM.si-CM from the xenograft cells when compared to ECs cultured on mock- or pSV-CM as determined by Ki-67 staining and MTT assays. Since Stat5 is a known target of IL-3, induces EC proliferation, and promotes neo-angiogenesis, we examined the role of pM.si in regulating IL-3-mediated Jak2/Stat5 signaling in ECs. Jak2/Stat5 suppression in ECs when cultured on pM.si-CM resulted in the decreased expression of integrin β1, Stat5, Bcl2 and Cyclin D2 at the transcription level as compared with ECs cultured on mock- and pSV-CM, as confirmed by RT-PCR analysis. Supplementation of recombinant human IL-3 counteracted and noticeably reversed pM.si-inhibited IL-3Rα, Jak2, p-Stat5 expression and proliferation of ECs, thereby confirming the role of MMP-2 in the regulation of α5β1/IL-3 mediated Jak2/Stat5 signaling. Immunoprecipitation and immunofluorescence studies revealed MMP-2 knockdown resulted in the decreased interaction of MMP-2 and integrin α5β1 in ECs, thereby implicating the downregulation of Jak2/Stat5 expression. Immunohistochemical analysis in tumor sections corroborated our in vitro studies, suggesting that the expression of p-Stat5 and Bcl2 are significantly decreased upon pM.si treatment compared to mock or pSV treatments. Our results suggest that MMP-2 knockdown by pM.si can lead to a significant inhibition of IL-3-induced proliferation of ECs and can be an effective anti-angiogenic therapy when treating patients with GBM in the future.

Citation Format: Dilip Rajasekhar Maddirela, Divya Kesanakurti, Chandramu Chetty, Jasti S. Rao. MMP-2 suppression inhibits α5β1 integrin and IL-3-mediated Stat5 activation and proliferation of endothelial 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 5100. doi:10.1158/1538-7445.AM2013-5100
Abstract 5114: Multi-organ metastatic capability of 4T1 mammary carcinoma cells after surgical resection of primary orthotopic tumor.
Authors: Zhang Yong , Zhang Nan , Hoffman Robert M. , Zhao Ming
Published: August 2014

We report an optically imageable orthotopic metastatic syngeneic model of 4T1 mouse breast cancer expressing red fluorescent protein (RFP). 4T1 cells, expressing RFP, were injected orthotopically into the right second mammary fat pad of BALB/c mice. The primary tumor was removed on day-18 after tumor implantation, when the average tumor volume reached approximate 500-600 mm3. At autopsy, the 4T1-RFP was found to have metastasized to numerous organs, when the mice were sacrificed 6-8 weeks after cell implantation, including the lung in 91% of the animals, the lymph nodes in 100% of the animals including axillary nodes, the liver in 91% of the animals, the bone in 42% of the animals and the brain in 25% of the animal. Detailed fluorescence analysis visualized extensive metastasis in the thoracic cavity and the lymphatic system. Large metastatic nodules in the lung involved most of the pulmonary parenchyma in all lobes. Lymph node metastasis was found mainly in the axillary area. In the liver, fluorescent macroscopic metastatic nodules were found under the capsule. The metastatic pattern in the model thus reflected clinical highly metastatic breast cancer and enables drug discovery for this disease.

Citation Format: Yong Zhang, Nan Zhang, Robert M. Hoffman, Ming Zhao. Multi-organ metastatic capability of 4T1 mammary carcinoma cells after surgical resection of primary orthotopic tumor. [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 5114. doi:10.1158/1538-7445.AM2013-5114
Abstract 5128: Upregulation of epsin in breast cancer and critical role of epsin in promoting cancer growth and metastasis.
Authors: Cai Xiaofeng , Pasula Satish , Chang Baojun , Hahn Scott , McManus John , Chen Hong
Published: August 2014

We have previously demonstrated that deficiency of epsins, a family of endocytic clathrin adaptor proteins, decreases tumor growth by enhancing VEGF signaling in endothelial cells and causing dysfunctional tumor angiogenesis. However, the role of epsin in cancer cells is unclear. Our preliminary data indicate that epsins are upregulated in various human cancers, including breast cancer, prostate cancer, ovary cancer, cervix cancer, bladder cancer, and esophagus cancer. Here, we determined the role of epsin in cancer cells in regulating breast cancer growth and metastasis. Increased epsin level was found in ER(+/-) and/or PR(+/-) human breast cancer cell lines including MDA-MB-231, BT-20, ZR751, MCF7 and T47D, and in mouse breast cancer cell lines, such as

168FARN and 4T1. The level of epsins was also considerably augmented in tumors derived from MMTV-PyMT spontaneous breast cancer mouse model at different progressive stages. We observed a correlative increase in epsin expression with cancer progression in MMTV-PyMT mice. We also generated stable epsin-deficient MDA-MB-231 and 4T1 cells. Epsin deficiency markedly inhibited cell proliferation and migration in vitro. Xenograft mouse models of epsin-deficient breast cancer cells revealed striking reduction in tumorigenesis and lung metastasis. Lung metastasis by tail-vein injection of epsin-deficient MDA-MB-231 cells was also decreased compared to controls. Mechanistically, we showed that epsin-deficiency induced morphological change from mesenchamal-like to epitheial-like in MDA-MB-231 cells, indicating a reversal of Epithelial-Mesenchymal Transition (EMT) to Mesenchymal-Epithelial-Transition (MET). Moreover, epsin-deficiency increased E-cadherin expression but decreased vimentin, and EMT inducing genes snail/slug expression in MDA-MB-231 cells. Epsin-deficiency also dramatically reduced cyclin D1 expression and Rb activation. Accordingly, elevated E-cadherin but attenuated vimentin, slug and cyclin D1 were observed in tumor tissues or lung tissues isolated from epsin-deficient MDA-MB-231 and 4T1 tumor models. Conversely, overexpression of epsin in MDA-MB-231 cells enhanced cyclin D1 and vimentin expression. Collectively, epsin promotes breast tumor growth and metastasis through coordinated regulation of cell cycle and EMT. Our study provides a basis for novel therapeutic targets for anti-cancer treatments.

Citation Format: Xiaofeng Cai, Satish Pasula, Baojun Chang, Scott Hahn, John McManus, Hong Chen. Upregulation of epsin in breast cancer and critical role of epsin in promoting cancer growth and metastasis. [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 5128. doi:10.1158/1538-7445.AM2013-5128
Abstract 5135: Analysis of formalin-fixed paraffin-embedded (FFPE) samples.
Authors: Stojanov Petar , Carter Scott L. , Rosshandler Ivan Imaz , Sivachenko Andrey , Salido Guadarrama Alberto , Vazquez Karla , Cordoba Sandra Romero , Cibulskis Kristian , Sougnez Carrie , Voet Douglas , Saksena Gordon , Lichtenstein Lee , Zou Lihua , Frazer Scott , Stewart Chip , Beroukhim Rameen , Meyerson Matthew , Lawrence Michael S. , Getz Gad
Published: August 2014

The availability of large numbers of formalin-fixed, paraffin-embedded (FFPE) tumor DNA samples, together with accurate and specific clinical annotations, can be a great resource for in-depth analysis of correlations of somatic and germline DNA alterations with clinical outcome. However, the effects of this tissue preservation technique on next-generation DNA sequencing technologies and downstream analyses are still being investigated. Furthermore, usually the archives of such FFPE samples do not include matched normal tissue, which complicates the process of identifying somatic alterations. We are in the process of analyzing lung adenocarcinoma, colorectal and prostate patient datasets, which contain both an FFPE and a fresh-frozen tumor together with a matched blood normal sample. The availability of both a frozen and an FFPE tumor sample enables us to compare the detected somatic point mutations and indels, taking into account the fact that these pairs are produced from different aliquots of DNA. This analysis includes calculating the power to detect variants in the FFPE sample given that they have been observed in the fresh frozen tumor, as well as the validation rate of clonal SNVs between the two samples. We are also developing a method for classification of SNVs as germline or somatic without a paired normal sample by taking advantage of the fact that most tumor samples contain a substantial fraction of normal cells. Because stromal contamination has a different effect on the allelic fraction of somatic vs. germline SNVs, we can apply algorithms to estimate tumor purity and absolute somatic copy numbers in order to distinguish the two types of events. We present preliminary results from these analyses.

Citation Format: Petar Stojanov, Scott L. Carter, Ivan Imaz Rosshandler, Andrey Sivachenko, Alberto Salido Guadarrama, Karla Vazquez, Sandra Romero Cordoba, Kristian Cibulskis, Carrie Sougnez, Douglas Voet, Gordon Saksena, Lee Lichtenstein, Lihua Zou, Scott Frazer, Chip Stewart, Rameen Beroukhim, Matthew Meyerson, Michael S. Lawrence, Gad Getz. Analysis of formalin-fixed paraffin-embedded (FFPE) samples. [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 5135. doi:10.1158/1538-7445.AM2013-5135
Abstract 5151: Integrative genomic analysis identifies cancer-testis antigen LEMD1 as a prognostic biomarker.
Authors: Park Jenny , Chiu Beatrice , Delaney Joe , Paschal Cate , Roche Daniel , Shima James , Umesh Anita , Wisotzkey Robert , Shekar Mamatha , Kupershmidt Ilya
Published: August 2014

The ability to molecularly distinguish disease states can improve prognostic resolution and therapeutic options. Cancer-testis antigens (CTA), expressed in germ cells of the testis and in a variety of cancers, are of particular interest for their potential use as targets for anti-cancer vaccines. We highlight the application of the NextBio platform, which integrates multiple datatypes such as RNA/miRNA expression, copy number variation, epigenetic changes, and next-generation sequencing with clinical attributes to identify and characterize novel disease associations in multiple myeloma.

Genomic and clinical data of 2,546 multiple myeloma patients obtained from public data sources were investigated in the NextBio platform. We initially analyzed the gene expression of various clinical subgroups to the median expression of all groups to identify genes significantly correlated to patient survival. We filtered for patients with advanced disease, defined as those with beta-2 microglobulin blood levels greater than 5.5mg/L, and identified LEMD1, a CTA, as positively correlated with survival (p=2.3E-6). We then analyzed the gene expression of LEMD1 in patients of all disease stages, and found those with increased expression of LEMD1 had improved survival compared to those who did not (p=9.4E-3). Patients with increased expression of LEMD1, however, had concomitant decreased expression in 14/20 CTAs of the MAGE family (p < 0.05), members of which were previously described as expressed in multiple myeloma patients with reduced survival.

A preliminary exploration of LEMD1 expression in other cancers found increased expression of LEMD1 to be significantly associated with improved survival in ovarian cancer (p=5.4E-3) but reduced survival in renal cancer (p=0.01). Further analysis of LEMD1 expression in normal tissues showed high expression of LEMD1 in spermatozoa and limited expression in 315 other tissue and cell types. As tissue specific antigens behind the blood-testis barrier, the expression of CTAs in cancer can potentially serve as immunogenic targets, and further investigation is warranted to explore their use in tumor immunotherapy.

Citation Format: Jenny Park, Beatrice Chiu, Joe Delaney, Cate Paschal, Daniel Roche, James Shima, Anita Umesh, Robert Wisotzkey, Mamatha Shekar, Ilya Kupershmidt. Integrative genomic analysis identifies cancer-testis antigen LEMD1 as a prognostic biomarker. [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 5151. doi:10.1158/1538-7445.AM2013-5151
Abstract 5161: Identification of epithelial stromal interaction 1 as a novel mediator of breast cancer cell invasion and metastasis downstream of Krüppel-like factor 8.
Authors: Li Tianshu , Lu Heng , Shen Chao , Lahiri Satadru , Watson Melissa , Mukherjee Debarati , Yu Lin , Zhao Jihe
Published: August 2014

Epithelial stromal interaction 1 (EPSTI1) is a recently identified novel gene that is highly upregulated in breast cancer cells when co-cultured with the stromal fibroblasts and in invasive breast carcinomas as compared with normal breast biopsies. The regulation and function of EPSTI1 are largely unknown. Our studies showed that EPSTI1 expression is upregulated by Kruppel-like factor 8 (KLF8), a transcriptional factor that is highly over-expressed in breast cancer and critical for cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition (EMT) and metastatic progression of breast cancer, by directly activating EPSTI1 promoter. Also, we demonstrated that EPSTI1 was necessary and sufficient to promote breast cancer cell invasiveness by genetically modifying EPSTI1 expression and Matrigel invasion assays. We also found that EPSTI1 could interact with valosin containing protein (VCP) and subsequently activate NF-kB nuclear function. Finally, our mouse mammary fat pad injection and tail vein injection experiments indicated that ectopic overexpression of EPSTI1 in MCF7 cells could enhance the tumor invasion and metastasis in vivo. These findings suggest that EPSTI1 is a novel target of KLF8 and plays a critical role in the regulation of breast cancer invasion and metastasis.

Citation Format: Tianshu Li, Heng Lu, Chao Shen, Satadru Lahiri, Melissa Watson, Debarati Mukherjee, Lin Yu, Jihe Zhao. Identification of epithelial stromal interaction 1 as a novel mediator of breast cancer cell invasion and metastasis downstream of Krüppel-like factor 8. [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 5161. doi:10.1158/1538-7445.AM2013-5161

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.