bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 442: RASA1 alteration promotes melanoma tumorigenesis
Authors: Sung Hyeran , Ding Li , Kanchi Krishna L. , Messina Jane L. , Sondak Vernon K. , James Mulé J. , Wilson Richard K. , Weber Jeffrey S. , Kim Minjung
Published: May 2015

Melanoma shows frequent Ras-Raf-MAPK pathway activation via frequent mutations in NRAS and BRAF. Novel mutations in other components of this pathway such as MEK1, MEK2, MAP3K5, and MAP3K9 have been reported recently by high-throughput sequencing efforts. In addition, NF1 (neurofibromatosis type 1), one of the Ras GTPase activating proteins (RasGAPs), has been shown to be mutated or suppressed in melanoma. However, importance of other RasGAPs has not been addressed in melanoma.

To obtain a comprehensive view of melanoma genomes, we conducted whole genome sequencing (WGS) of 15 metastatic melanomas and matched normal PBMC genomes from 13 melanoma patients. All melanoma genomes from these 13 patients contained at least one mutation in genes of Ras-Raf-MAPK pathway (MAPK1, MAP3K1, MAP4K2, MAP3K14, NRAS, and BRAF). Our whole genome analyses also identified two novel somatic missense mutations (p.Tyr472His and p.Leu481Phe) in or around the PH domain in RASA1 gene. RASA1 (RAS p21 protein activator (GTPase activating protein) 1, also called as p120RasGAP) is a GTPase activation protein that negatively regulates Ras by catalyzing the hydrolysis of active Ras-GTP to inactive Ras-GDP. In this study, we addressed possible roles of RASA1 in melanoma tumorigenesis. To determine RASA1 level in melanocytic lesions, immunohistochemical analysis of RASA1 on human melanoma tissue microarray (TMA) containing nevi (n=34), primary melanomas (n=63), lymph node metastasis (n=35), and distal metastasis (n=29) was performed. This analysis has shown the inverse correlation of RASA1 expression to melanoma progression. Functionally, ectopic expression of RASA1 mutant increased soft-agar colony formation and tumor growth while that of wild-type RASA1 decreased. shRNA-mediated knock down of RASA1 promoted soft-agar colony formation and invasion of melanocytes and melanoma cells with BRAF mutation as a consequence of the elevated Ras activity.

Here, we established the functional significance of novel somatic missense mutations in RASA1 identified in our WGS study. Collectively, these data implicated that RASA1 inactivation by loss of expression and/or mutation may promote melanoma tumorigenesis.

Citation Format: Hyeran Sung, Li Ding, Krishna L. Kanchi, Jane L. Messina, Vernon K. Sondak, Mulé J. James, Richard K. Wilson, Jeffrey S. Weber, Minjung Kim. RASA1 alteration promotes melanoma tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 442. doi:10.1158/1538-7445.AM2014-442
Abstract 4420: Matriptase-mediated proteolytic regulation of biological activity and spatial distribution of PDGF-D
Authors: Huang Wei , Kim Hyeong-Reh C.
Published: May 2015

The oncogenic roles of platelet-derived growth factor D (PDGF-D) and its proteolytic activator, matriptase, have been strongly implicated in human prostate cancer. The latent full-length PDGF-D consists of N-terminal CUB domain, C-terminal growth factor domain, and hinge region in between. Matriptase processes the full-length PDGF-D dimer (FL-D) into PDGF-D growth factor domain dimer (GFD-D) in a step-wise manner, involving generation of hemidimer (HD), which is an intermediate product containing one full-length PDGF-D subunit and one growth factor domain subunit. In this study, we showed that HD acts as a dominant-negative ligand of PDGF/PDGFR signaling in fibroblasts whereas it has its own unique function in pre-osteoclasts. The regulation of PDGF-D's activity by matriptase is biphasic as demonstrated by the event that active PDGF-D GFD-D can be further cleaved into a smaller and yet inactive form if the enzymatic action of matriptase persists. By mutagenesis analyses, we identified amino acid residues R340R341GR343A at the C-terminus of PDGF-D growth factor domain to be the matriptase cleavage site through which PDGF-D is inactivated. Based on the sequence analysis in comparison with the published crystal structure of PDGF-B, we suggest that R340R341GR343A is within the loop III of PDGF-D growth factor domain, a critical domain for its binding to the PDGF receptor. When the positively charged arginine residues of R340R341GR343A were mutated to alanine residues, PDGF-D GFD-D lost its biological activity in activating β-PDGFR. Further, we found that proteolytic processing of latent PDGF-D to remove CUB domain and hinge region is tightly linked with the storage of PDGF-D GFD-D within extracellular matrix (ECM) in vitro. These results indicate the increased affinity to the ECM from FL-D, HD, to GFD-D and also suggest that CUB domain and hinge region of PDGF-D prevent the sequestration of PDGF-D in ECM. Interestingly, the matriptase cleavage site R340R341GR343A within the growth factor domain is also critical for PDGF-D association with ECM. Taken together, the present study reports structural elements crucial to the biologically function of PDGF-D and its association with ECM, suggesting that proteolytic processing is a crucial mechanism for PDGF-D to establish its tissue gradient.

Citation Format: Wei Huang, Hyeong-Reh C. Kim. Matriptase-mediated proteolytic regulation of biological activity and spatial distribution of PDGF-D. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4420. doi:10.1158/1538-7445.AM2014-4420
Abstract 4422: A Hippo-independent pathway modulates YAP-induced resistance of cancer cells to antitubulin drugs
Authors: Zhao Yulei , Khanal Prem , Yang Xiaolong
Published: May 2015

Antitubulin drugs have been widely used for the treatment of various human cancers including breast and lung cancer, etc. Although these drugs are effective as first-line treatment of cancers, many cancer patients are either intrinsically resistant or later develop resistant to drugs, which is one of the major obstacles for successful cancer treatment. Therefore, understanding the molecular mechanism underlying antitubulin drug resistance is critical for future prognosis of cancer treatment and development of novel therapeutic drugs for cancer treatment. The Hippo pathway is an emerging signaling pathway that plays important roles in various biological processes including organ size control, tumorigenesis, stem cell differentiation and renewal, etc. Although tremendous progress has been made toward our understanding of the roles of the Hippo pathway in cancer development, if and how is its role in drug resistance remains largely unknown. Recently, we and others have provided convincing evidence that some core components of the Hippo pathway are involved in the resistance of cancer cells to antitubulin drug Taxol. However, the signaling pathways or genes mediating their effects in chemotherapeutic drug response remain to be identified. In this study, we have discovered that Hippo core component YAP oncoprotein and transcriptional co-activitor is specifically phosphorylated only after treatment of cancer cells with antitubulin drugs. Most significantly, we have identified a Hippo-independent pathway in which activated Cdk1 can directly phosphorylate YAP, which subsequently inactivates YAP function in transactivation and anti-apoptosis during antitubulin drug treatment. These findings have significant implication not only in future use of YAP as a prognostic marker in predicting antitubulin drug response in patients but also provide a novel approach for future targeting and inactivating YAP by activating its phosphorylation for treatment of antitublin drug resistant cancer patients caused by YAP activation.

Citation Format: Yulei Zhao, Prem Khanal, Xiaolong Yang. A Hippo-independent pathway modulates YAP-induced resistance of cancer cells to antitubulin drugs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4422. doi:10.1158/1538-7445.AM2014-4422
Abstract 4425: Drug resistance via feedback activation of Stat3 in oncogene-addicted cancer cells
Authors: Lee Ho-June , Zhuang Guanglei , Cao Yi , Du Pan , Kim Hyo-Jin , Settleman Jeff
Published: May 2015

Targeted therapy directed against ‘oncogene-addicted’ cancers can produce dramatic responses that are invariably of limited durability due to the emergence of subpopulations of drug-resistant cells. We found that many oncogene-addicted cancer cells treated with pathway-targeted drugs engage a positive feedback loop leading to activation of Stat3, which promotes cell survival, consequently limiting the overall drug response. This was observed in cancer cells driven by diverse mutationally-activated kinases, including EGFR, HER2, ALK, and MET, as well as mutant KRAS. We determined that inhibition of MEK signaling specifically promotes Stat3 engagement. MEK inhibition led to the autocrine-driven activation of Stat3 via the FGF receptor and JAK family kinases, and pharmacological inhibition of MEK together with JAK and FGFR effectively promoted tumor regression in vivo. These findings suggest that inhibition of a Stat3 feedback loop may enhance the response to a broad spectrum of drugs that target pathways of oncogene addiction.

Citation Format: Ho-June Lee, Guanglei Zhuang, Yi Cao, Pan Du, Hyo-Jin Kim, Jeff Settleman. Drug resistance via feedback activation of Stat3 in oncogene-addicted cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4425. doi:10.1158/1538-7445.AM2014-4425
Abstract 4426: Tumor suppressive effects of wild-type Hras on oncogenic Kras-driven pancreatic tumorigenesis
Authors: Weyandt Jamie D. , Counter Christopher M.
Published: May 2015

The RAS proteins are a group of small GTPases that can become constitutively activated by point mutations that are found in a quarter or more of all cancer patients, particularly in pancreatic cancer, in which over 90% of patients have an activating KRAS mutation. There are three well-characterized RAS protein family members: HRAS, NRAS, and KRAS, the latter of which is alternatively spliced at the C-terminus into two proteins, KRAS4A and KRAS4B. The RAS proteins are all nearly identical at their N-termini and core effector binding domains. However, they have divergent C-terminal membrane-binding regions that impart both different subcellular localization and subtle changes in signaling. While oncogenic RAS is well established to promote cancer, recent work has suggested that wild-type RAS proteins also participate in tumorigenesis. In this regard, we previously found that wild-type HRAS is activated downstream of oncogenic KRAS, which promoted tumor growth of human pancreatic cancer cell lines. To examine the role of wild-type Hras during de novo pancreatic tumor development, we tested whether knockout of the wild-type Hras gene altered tumorigenesis in oncogenic Kras-driven mice models of pancreatic cancer. Specifically, Hras homozygous null mice (Hras-/-) were crossed into a Pdx-Cre;LSL-KRasG12D/+ background in the absence or presence of an additional mutant p53 allele (Trp53R172H/+) to induce early and late pancreatic cancer, respectively. Surprisingly, loss of Hras led to an increase in early pancreatic lesions and reduced survival in the model of late disease. Since HRAS is activated downstream of oncogenic KRAS, and high oncogenic signaling can induce a senescent growth arrest, we tested and found that suppressing senescence by mutating both alleles of Trp53 ameliorated the survival difference between wild-type and null Hras mice. We thus hypothesize that wild-type Hras amplifies oncogenic Kras signaling, leading to the growth arrest of senescence and thereby inhibit early tumorigenesis. However, once senescence is suppressed later in tumorigenesis, wild-type RAS proteins instead promote more malignant phenotypes.

Citation Format: Jamie D. Weyandt, Christopher M. Counter. Tumor suppressive effects of wild-type Hras on oncogenic Kras-driven pancreatic tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4426. doi:10.1158/1538-7445.AM2014-4426
Abstract 4427: Copper is required for oncogenic BRAF signaling and tumorigenesis
Authors: Brady Donita C. , Crowe Matthew S. , Turski Michelle L. , Hobbs G Aaron , Chaikuad Apirat , Knapp Stefan , Campbell Sharon L. , Thiele Dennis J. , Counter Christopher M.
Published: May 2015

The BRAF serine/threonine kinase is mutated, typically at V600, to induce an active oncogenic state in a large fraction of melanoma, thyroid cancers, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers. BRAFV600E in turn activates the kinases MEK1 and MEK2, which stimulates the MAPK pathway to promote cancer. Excitingly, ATP inhibitors of oncogenic BRAF and MEK provide a survival advantage in metastatic melanoma, an effect enhanced by combining the two inhibitors. Thus, combining multiple approaches to inhibit MAPK signaling holds great promise for the treatment of BRAF mutation-positive cancers, especially in terms of overcoming resistance. In this regard, we previously found that copper (Cu) influx enhanced MEK1 phosphorylation of its substrates ERK1/2 through a Cu-MEK1 interaction. We show here that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced MAPK signaling and oncogenic BRAFV600E-mediated tumorigenesis, which was rescued by expressing activated MEK5 engineered to phosphorylate ERK1/2 or activated ERK2. Importantly, Cu chelators used in the treatment of Wilson disease reduced tumor growth of not only BRAFV600E-transformed cells but also cells resistant to a BRAF inhibitor. Taken together, these results suggest that Cu-chelation therapy could be repurposed for the treatment of BRAFV600E mutation-positive cancers.

Citation Format: Donita C. Brady, Matthew S. Crowe, Michelle L. Turski, G Aaron Hobbs, Apirat Chaikuad, Stefan Knapp, Sharon L. Campbell, Dennis J. Thiele, Christopher M. Counter. Copper is required for oncogenic BRAF signaling and tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4427. doi:10.1158/1538-7445.AM2014-4427
Abstract 4428: Oncogenic ERBB3 mutations in human cancers
Authors: Jaiswal Bijay S. , Kljavin Noelyn M. , Stawiski Eric , Durinck Steffen , Chaudhuri Subhra , Eigenbrot Charles , Schaefer Gabriele , Sauvage Frederic J. de , Seshagiri Somasekar
Published: May 2015

The human epidermal growth factor receptor (HER) family of tyrosine kinases are deregulated in multiple cancers either through amplification, overexpression or mutation. ERBB3/HER3, the only ERBB family member with an impaired kinase domain, upon ligand binding heterodimerizes with ERBB2 to promote signaling. While amplification and overexpression of ERBB3 is observed in some cancers, occurrence and relevance of ERBB3 somatic mutations in oncogenesis is not established. Here we report the identification of ERBB3 somatic mutations in ∼11% of colon and gastric cancers. We found that the ERBB3 mutants together with ERBB2 promote oncogenic signaling and transformed colonic and breast epithelial cells in a ligand independent manner. Further, we found that multiple target therapeutics that acts on ERBB3, ERBB2 or their downstream signaling components are effective in blocking ERBB3-mutant mediated oncogenic signaling and disease progression in vivo. Identification of activating ERBB3 mutations along with the established contribution of ERBB3 to acquired resistance to EGFR/ERBB2-targeted drugs provides a rational to therapeutically target ERBB3.

Citation Format: Bijay S. Jaiswal, Noelyn M. Kljavin, Eric Stawiski, Steffen Durinck, Subhra Chaudhuri, Charles Eigenbrot, Gabriele Schaefer, Frederic J. de Sauvage, Somasekar Seshagiri. Oncogenic ERBB3 mutations in human cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4428. doi:10.1158/1538-7445.AM2014-4428
Abstract 443: Synergistic inhibition of HER2 positive breast cancer by triptolide and lapatinib
Authors: Chalugun Paweena , Shim Joong Sup , Korangath Preethi C. , Sukumar Saraswati , Liu Jun O.
Published: May 2015

HER2-positive breast cancers are poorly differentiated and have a higher potential for metastasis. Although trastuzumab and lapatinib have been used for the treatment of HER2-positive breast cancer, resistance to both drugs has dampened the long-term benefit of those drugs. As cancer has multiple genetic aberrations and can readily develop resistance to individual drugs with specific targets, we employed synergistic drug combination as a strategy to overcome drug resistance and to enhance the efficacy of existing drugs. From a screen of the Johns Hopkins Drug Library, we identified triptolide as a strong synergistic hit with lapatinib in HCC1954, a drug-resistant HER2-positive breast cancer cell line. The combination of lapatinib and nontoxic doses of triptolide synergistically inhibited the HER2 signaling pathway. We found that lapatinib increased both mRNA expression and the half-life of HER2 protein, whereas triptolide countered the effects of lapatinib by downregulating the expression of HER2, leading to synergistic inhibition of the HER2 signaling pathway. The synergy in cell proliferation was observed in all HER2-positive breast cancer cell lines regardless of drug resistance status, but was absent in HER2-negative ones. In mouse xenograft models, the combination of lapatinib and triptolide at their minimum effective doses almost completely inhibited the growth of the drug-resistant HER2-positive breast cancer cells. Together, these findings suggest that the combination of triptolide and lapatinib could be a promising new therapeutic regimen for drug-resistant HER2-positive breast cancer.

Citation Format: Paweena Chalugun, Joong Sup Shim, Preethi C. Korangath, Saraswati Sukumar, Jun O. Liu. Synergistic inhibition of HER2 positive breast cancer by triptolide and lapatinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 443. doi:10.1158/1538-7445.AM2014-443
Abstract 4431: N-terminal portion of β-catenin is important in ISGylation of β-catenin by 90K glycoprotein in colorectal cancer cells
Authors: Yoon Somy , Bae Jeong A , Kim Hangun , Moon Kyung-Sub , Kim Kyung Keun
Published: May 2015

β-catenin is a major transducer in Wnt signaling pathway, which is highly expressed in colorectal and other cancers. Previously, we have shown that β-catenin is down-regulated in colorectal cancer cells by glycoprotein, 90K, through ISGylation-dependent ubiquitination. The aim of this study is to further clarify a novel mechanism of β-catenin down-regulation pathway by 90K glycoprotein. To identify ISGylation site of β-catenin, deletion mutants of β-catenin lacking N- or C-terminal domain were employed. As only two lysine residues, K19 and K49, are reside in N-terminus of β-catenin, we sequenced cDNA of β- catenin from paired human colorectal cancer tissues to find the mutation and generated arginine point-mutation. As a result, there is no mutation on K19 and/or K49 in examined specimens and did not affect the 90K-mediated ISGylational degradation. To rule out the possibility of involving non-lysine residue in the ISG15 conjugation, such as serine or threonine, we constructed several deletion mutants and found that none are responsible for the ISG15 conjugation. Now, N-terminal portion of β-catenin is under screening for possible interactions with putative E3 ligases, and further detailed studies delineating the complex formation among ISG15 and β-catenin/E3 ligases are under way.

Citation Format: Somy Yoon, Jeong A Bae, Hangun Kim, Kyung-Sub Moon, Kyung Keun Kim. N-terminal portion of β-catenin is important in ISGylation of β-catenin by 90K glycoprotein in colorectal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4431. doi:10.1158/1538-7445.AM2014-4431
Abstract 4432: The involvement of suppressor of cytokine signaling 6 (SOCS6) in the ECS E3 ubiquitin ligase complex
Authors: Chen Shu-Chuan , Lin Huan-Yu , Lin Shiu-Ting , Wang Mei-Jung , Chen Jeou-Yuan
Published: May 2015

SOCS proteins were initially identified as the suppressors of cytokine signaling. There are eight members in the SOCS family, each containing a central SH2 domain and a carboxyl-terminal SOCS box motif. Among the SOCS family proteins, CIS and SOCS1-3 have been shown to be expressed at elevated levels upon cytokine treatment, and the up-regulated proteins can then turn around to suppress the cytokine signaling through recruiting the phosphorylated signaling intermediates for ubiquitylation-mediated protein degradation, leading to the blockade of the signaling pathway. However, the involvement of other SOCS family proteins, including SOCS6, in protein ubiquitylation remains to be addressed. We have previously shown that biallelic inactivation of SOCS6 is a frequent event in human gastric cancer, and that overexpression of SOCS6 inhibits cell growth as well as colony formation, suggesting a potential role of SOCS6 as a tumor suppressor. We further demonstrated that SOCS6 is targeted to the mitochondria, and induces mitochondrial fragmentation accompanied with intrinsic apoptosis. In this study, we explored the possible linkage of SOCS6 to the formation of an E3 ubiquitin ligase complex. By GST-pulldown and immunoprecipitation assays, we showed that SOCS6 interacted with Elongin B/C and Cullin 5. At steady state, SOCS6 is expressed at relatively low level, but its level is increased in the presence of proteasome inhibitor MG132. In addition, ectopic expression of Elongin B/C and/or Cullin 5 also led to the stabilization of SOCS6 protein. We also showed that formation of an intact Elongin B/C-Cullin 5-SOCS (ECS) E3 ligase complex is important for SOCS6-mediated mitochondrial fragmentation. Mutations of the conserved Leu500 and Cys504 residues in the BC-box located in SOCS box motif yielded a SOCS6 mutant that not only lost the ability to interact with Elongin B/C but also failed to induce mitochondrial fragmentation and apoptosis. Most importantly, ablation of Elongin C activity protected cells from SOCS6-mediated mitochondrial fission. These data suggest an important role of SOCS6 in modulating mitochondrial dynamics mediated through ECS E3 ubiquitin ligase complex activity. We have also searched for substrates of SOCS6-mediated ECS E3 ubiquitin ligase complex by immunoprecipitation using an anti-K-ϵ-GG antibody followed by proteome study. The characterization of the ubiquitylated candidate proteins will be further discussed.

Note: This abstract was not presented at the meeting.

Citation Format: Shu-Chuan Chen, Huan-Yu Lin, Shiu-Ting Lin, Mei-Jung Wang, Jeou-Yuan Chen. The involvement of suppressor of cytokine signaling 6 (SOCS6) in the ECS E3 ubiquitin ligase complex. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4432. doi:10.1158/1538-7445.AM2014-4432
Abstract 4443: The tumor suppressive small GTPase DiRas1 binds the RhoGEF SmgGDS and antagonizes RhoA activation
Authors: Hauser Andrew D. , Barr Kristen M. , Frei Anne C. , Gonyo Patrick , Lorimer Ellen L. , Williams Carol L. , Bergom Carmen
Published: May 2015

A number of malignancies are promoted by the activation of Rho GTPases. Regulators of RhoA activity, including the RhoGEF, SmgGDS, can also promote cancer development. Both RhoA and SmgGDS have been shown to enhance NF-ĸB activation in a number of tumors. Utilizing mass spectrometry, we identified the tumor suppressive small GTPase DiRas1 as a novel binding partner for SmgGDS. DiRas1 is a tumor suppressor in malignant gliomas and esophageal squamous cell carcinomas. Examining DiRas gene expression using the ONCOMINE mRNA microarray database (www.oncomine.org) revealed that DIRAS1 and DIRAS2 are markedly decreased in central nervous system tumors, including anaplastic astrocytomas and GBMs, when compared to normal tissue. The role of DiRas1 in other cancers is largely undetermined, and the ways in which DiRas1 may antagonize pro-oncogenic small GTPase signaling is largely unknown. To define how DiRas1 expression may affect SmgGDS functions, we tested the association of DiRas family proteins with SmgGDS. SmgGDS robustly bound wildtype DiRas1, and its closely-related family member DiRas2, but only weakly bound DiRas3 in vitro and in cell lines. In addition, DiRas1, DiRas2, or DiRas3 protein expression decreased proliferation of HEK293T cells. DiRas1 expression could promote less activated RhoA, as a low level of DiRas1 expression caused markedly decreased RhoA-SmgGDS binding in a number of cell lines. DiRas1 expression also abrogated SmgGDS- and RhoA-mediated NF-ĸB activation in a concentration-dependent manner. SmgGDS may exhibit GEF activity toward DiRas1, as a dominant negative DiRas1 (S21N) binds to SmgGDS more than the wildtype protein. However, DiRas1 is predominantly GTP-bound in cells, likely due to a high rate of guanine nucleotide dissociation and low intrinsic GTPase activity. Thus, DiRas1 expression may in part promote tumor suppression by non-productively binding SmgGDS, resulting in less RhoA activity. Taken together, these results suggest that the tumor suppressive small GTPase DiRas1 can antagonize RhoA signaling by competing for binding of the RhoGEF SmgGDS. Further characterizing these actions may lead to novel therapeutic targeting of RhoA activation in cancer malignancies.

Citation Format: Andrew D. Hauser, Kristen M. Barr, Anne C. Frei, Patrick Gonyo, Ellen L. Lorimer, Carol L. Williams, Carmen Bergom. The tumor suppressive small GTPase DiRas1 binds the RhoGEF SmgGDS and antagonizes RhoA activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4443. doi:10.1158/1538-7445.AM2014-4443
Abstract 4444: The role of small GTPase Rab7 in the secretion of extracellular microvesicles by head and neck squamous cells carcinoma
Authors: Giudice Fernanda S. , Rodrigues Bruna R. , Dias Marcos V. S. , Baptistella Antuani R. , Lacerda Tonielli Cristina S. , Kowalski Luiz P. , Martins Vilma R.
Published: May 2015

Head and neck squamous cell carcinoma (HNSCC) is an epithelial malignant tumor that shows its highest incidence in the upper aerodigestive tract and is considered one of the six most common malignancies worldwide. Among the processes involved in the poor prognosis of HNSCC, local invasion and subsequent metastasis are the most important events. Recent evidences have shown that metastasis can be the result of extracellular microvesicles (MVEs) being released from the tumor cells to the blood or lymphatic system. The molecular mechanisms involved in MVEs biogenesis and secretion are, however, poorly understood. Herein, we have performed a screen targeting for the components of MVEs biogenesis in three HNSCC cell lines (SCC-9, Cal27 and FaDu) through western blotting. MVEs were quantified using Nanoparticle Tracking Analysis (NTA - NanoSight®) and the lysosomal compartment was followed in the cell lines through immunofluorescence. Comparing the three cell lines, FaDu cells presented higher expression of proteins responsible for MVEs formation (p<0.05), such as Flotilin-1, Tsg101, VPS36 and also Rab7, a small GTPase that directs vesicles to lysosomes. However, FaDu cells secreted less MVEs than SCC-9 and Cal27. Immunofluorescence experiments showed the presence of numerous perinuclear clusters positive to LAMP-1 (lysosome marker) in FaDu cells, while SCC-9 and Cal27 showed LAMP1-positive organelles dispersed throughout their cytoplasms. Taken together, the results highlight a role for Rab7 protein in the negative regulation of MVEs secretion by HNSCC cell lines, which may provide important information related to tumor progression, treatment response, and especially the development of metastasis.

Supported by São Paulo Research Foundation (FAPESP)

Citation Format: Fernanda S. Giudice, Bruna R. Rodrigues, Marcos V. S. Dias, Antuani R. Baptistella, Tonielli Cristina S. Lacerda, Luiz P. Kowalski, Vilma R. Martins. The role of small GTPase Rab7 in the secretion of extracellular microvesicles by head and neck squamous cells carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4444. doi:10.1158/1538-7445.AM2014-4444
Abstract 4456: Aurora-A is a downstream target of RAS and forms a positive feedback regulation loop with NF-κB in non-small cell lung cancer
Authors: Kim Donghwa , Kanai Masayuki , Zheng Xiangqian , Zheng Dali , Coppola Domenico , Cheng Jin Q.
Published: May 2015

Previous studies have shown that inhibition of Aurora-A reduces mutant RAS oncogenic activity and that both Aurora-A and RAS activate NF-κB pathway through phosphorylation of IkBa and upregulation of GSKa, respectively. Here we show that Aurora-A expression and kinase activity are regulated by activating mutation of KRAS. Blockage of NF-κB by IκB-S32/36A abrogated KRAS-induced Aurora-A expression and kinase activity. Furthermore, we demonstrated that NF-κB directly bound to Aurora-A promoter and induces Aurora-A transcription. In addition, depletion of Aurora-A by siRNA and pharmacological inhibitor MLN8237 suppresses RAS-induced NF-κB activity and selectively induces cell death in KRAS mutant NSCLC cells. We also show that Aurora-A is induced by nicotine and is frequently overexpressed in NSCLCs. Overexpression of Aurora-A is associated with tobacco smoke and poor prognosis in lung adenocarcinoma but not squamous lung cancer. These data suggest that Aurora-A is regulated by RAS through NF-κB and that the feedback regulation loop between Aurora-A and NF-κB could play a pivotal role in NSCLC.

Citation Format: Donghwa Kim, Masayuki Kanai, Xiangqian Zheng, Dali Zheng, Domenico Coppola, Jin Q. Cheng. Aurora-A is a downstream target of RAS and forms a positive feedback regulation loop with NF-κB in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4456. doi:10.1158/1538-7445.AM2014-4456
Abstract 4457: Regulation of GRM1 by estrogen receptor in breast cancer
Authors: Dolfi Sonia C. , Mehta Madhura S. , Kornblum David , Boughton Andrew , Rahim Hussein , Medina Daniel , Hirshfield Kim M.
Published: May 2015

Metabotropic glutamate receptor 1 (GRM1), first recognized as an important factor in the central nervous system, has more recently been implicated in melanomagenesis and breast cancer pathogenesis. Oncogenic mediators of GRM1-associated effects include both MAPK and AKT signaling cascades. Modulation of GRM1 activity in melanoma and estrogen receptor negative (ER-) breast cancer cells inhibits anchorage independent growth, migration, proliferation, and/or downstream activation of Akt and ERK. Data from our laboratory show that a significantly higher fraction of breast tumors express GRM1 compared to normal breast tissue, and GRM1 expression is more highly correlated with ER positivity. Moreover, higher GRM1 expression in human ER+ breast cancers treated with the anti-estrogen tamoxifen associates with a shorter distant metastasis free survival as compared to low expressors further supporting a critical role in ER+ breast cancer.

We hypothesize that ER has both genomic and non-genomic effects on GRM1. We evaluated the effect of hormonal treatment on GRM1 protein expression in ER+ MCF7 and ER- MDA-MB-231 breast cancer cells. Estradiol increases GRM1 expression, and this effect is abrogated by the anti-estrogen tamoxifen in MCF7 but not in MDA-MB-231 cells. These genomic effects may be exerted through three putative ER binding sites within the GRM1 promoter. We have confirmed that ER binds to one of these sites by chromatin immunoprecipitation, further supported by ChiPSeq data. GRM1 promoter activity is increased by estradiol treatment using a luciferase reporter assay validating transcriptional activation by ER. To support a non-genomic role for ER with GRM1, we have shown that ER physically interacts with GRM1 in breast cancer cells by in vitro proximity ligation assay and by co-immunoprecipitation in immortalized mouse mammary epithelial cells (iMMECs) stably expressing GRM1. Stable introduction of GRM1 into iMMECs, which lack endogenous GRM1, reduces sensitivity to tamoxifen as compared to control iMMECs. In MCF7 cells, the combination of tamoxifen with GRM1 modulators riluzole or BAY 36-7620 produces synergistic effects.

As estradiol activates MAPK signaling in breast cancer cells, we hypothesize that this may be mediated by both the physical interaction of GRM1 and ER and the indirect effects on GRM1 transcription. Results of these studies suggest that GRM1 may be a valuable therapeutic target in ER+ breast tumors.

Citation Format: Sonia C. Dolfi, Madhura S. Mehta, David Kornblum, Andrew Boughton, Hussein Rahim, Daniel Medina, Kim M. Hirshfield. Regulation of GRM1 by estrogen receptor in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4457. doi:10.1158/1538-7445.AM2014-4457
Abstract 4473: Development of ceramide liposomes for tumor interstitial and vascular drug targeting
Authors: Cheng Shaohua , Crall William , Nguyen Bryan , Dang Charles , Alkholief Musaed , Campbell Robert B.
Published: May 2015

Background: Interstitial and vascular targeting with cytotoxic agents has proven to be an effective approach for cancer therapy (1). Ceramide, a sphingolipid, plays an important role in apoptosis, regulation of differentiation, growth suppression, and in cell senescence (2). Liposomes containing ceramide exert an anti-proliferative effect against the growth of tumor cells in vitro (3). In this study, we attempted to apply a rational approach to the development of ceramide liposomes for tumor targeting.

Methods: We first determined the relative amounts of ceramide produced by cells normally found in the tumor interstitial matrix (ZR-75-1- human breast cancer cells), and by those lining tumor endothelia (HMEC-1 -human microvascular endothelial cells). For this experiment we measured sphingomyelinase activity. We next determined the cytotoxic effects of various liposome systems containing 0 to 20% ceramide (C6:1) content. The optimal ceramide content was determined from this study, and 2-methoxyestradiol (2-ME) was loaded in the optimal ceramide liposome preparation type. We next evaluated cytotoxic effects of the drug-loaded liposomes compared to ceramide liposomes alone.

Results: ZR-75-1 cells showed a higher level of sphingomyelinase activity (4.4 fold) as compared to that of HMEC-1. The 20% ceramide formulation demonstrated the most significant anti-proliferative effect, regardless of the cell line. The cytotoxic drug effects were favorably influenced by the ceramide content. Finally, preliminary studies suggest that the overall effectiveness of ceramide liposomes against the growth of proliferating tumor cells correlated with the sphingomyelinase activity expressed by the target cells.

Conclusion: Future studies will determine whether sphingomyelinase activity can be used to determine the population of cells (i.e., interstitial vs vascular) most likely to benefit from ceramide (cytotoxic) drug-loaded liposomes as a therapeutic approach.

Citation Format: Shaohua Cheng, William Crall, Bryan Nguyen, Charles Dang, Musaed Alkholief, Robert B. Campbell. Development of ceramide liposomes for tumor interstitial and vascular drug targeting. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4473. doi:10.1158/1538-7445.AM2014-4473
Abstract 4478: Biopharmaceutic studies of novel nucleoside transporter inhibitor prodrugs that can protect against nucleoside drug-induced mitochondrial toxicity
Authors: Sun Shan , Playa Hilaire C. , Deokar Hemantkumar S. , Lin Wenwei , Buolamwini John K.
Published: May 2015

Nucleoside analog antimetabolites are used as chemotherapy agents to treat cancer by interfering with DNA biosynthesis and therefore cell division and the growth of tumors. However, nucleoside drugs, such as the nucleoside reverse transcriptase inhibitor antiviral agents and gemcitabine, a drug approved for use against various solid tumors, have been found to cause severe mitochondrial toxicities that can limit their use. Nucleoside analogs cause toxicity by inhibiting mitochondrial DNA (mtDNA) synthesis. Nucleoside drugs can be transported from cytoplasm into mitochondrial by mitochondrial membrane nucleoside transporters (NTs) where they form the active triphosphate analogs and incorporate into mtDNA and inhibit mtDNA polymerase, Pol γ. Currently, there is no proven general treatment or prevention strategy for nucleoside drug-induced mitochondrial toxicity. Thus, the development of innovative approaches to preempt or treat mitochondrial toxicity will be an important advance in the field of nucleoside drug therapy.

In our research to overcome the mitochondrial toxicity, we developed a set of novel nucleoside transporter inhibitor (NTI) prodrugs that lack of NT inhibition but can release active drugs intracellularly to inhibit mitochondrial nucleoside uptake. In vitro biopharmaceutics studies of cell entry, in-cell hydrolysis profiles, and metabolic stability in human liver S9 faction and human plasma were carried out on a set of novel prodrugs that have shown protection against nucleoside drug-induced mitochondrial toxicity. Financial support by NIH grant RO1GM10405 is acknowledged.

Citation Format: Shan Sun, Hilaire C. Playa, Hemantkumar S. Deokar, Wenwei Lin, John K. Buolamwini. Biopharmaceutic studies of novel nucleoside transporter inhibitor prodrugs that can protect against nucleoside drug-induced mitochondrial toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2014-4478
Abstract 4486: Stromal depletion by a docetaxel nanoparticle for enhanced therapy of breast and pancreatic cancer
Authors: Ernsting Mark , Murakami Mami , Undzys Elijus , Li Shyh-Dar
Published: May 2015

Tumor stroma consists of genetically normal cells that express abnormal phenotype in the tumor microenviroment, and are drivers of tumor cell proliferation and migration processes. Stromal cells can represent a significant fraction of tumor mass, and represent a barrier to drug delivery, given that stromal fibroblasts exert contractile forces which reduce vascular perfusion and increase interstitial fluid pressure. In the field, the stroma is widely recognized as a target for therapy, both to normalize the reactive microenvironment, and to reduce drug delivery barriers, but with limited clinical success.

We have developed a nanoparticle delivery system for docetaxel (Cellax), a conjugate of carboxymethylcellulose and PEG, which condenses into well defined 120 nm particles suitable for intravenous administration. In addition to enhanced pharmacokinetics, 5-10X improved tumor uptake, and enhanced anti-tumor efficacy compared to approved taxanes such as native DTX (Taxotere) and nab-PTX (an albumin-paclitaxel nano-formulation, Abraxane), we have observed an anti-stromal effect in breast and pancreatic models. In a 4T1 orthotopic breast cancer model in mice, 85% of Cellax particles were taken up by cancer-associated fibroblasts (CAF), a major component of stroma. Further, in 4T1 and MDA-MB-231 breast models, Cellax treatments reduced CAF by 82 and 70% respectively, while native DTX and Nab-paclitaxel exerted no anti-stromal activity. Concomitant effects were ∼70-fold increased perfusion, ∼3-fold decreased interstitial fluid pressure (IFP), and a 7-24 fold reduction in lung metastases. In a primary human pancreatic xenograft model (OCIP19), we measured a 50% reduction in αSMA+ cells (activated pancreatic stellate cells) and macrophage within 1 day of Cellax treatment, while nab-PTX only decreased the macrophage population 3-7 days post therapy. In addition, vascular perfusion in the Cellax treated pancreatic tumor was increased 12X relative to nab-PTX and control treatment groups, with significantly reduced metastases. Preliminary mechanistic studies indicate that albumin absorbs to Cellax particles, promoting interaction with SPARC-expressing stromal cells, leading to stroma-specific activity.

Citation Format: Mark Ernsting, Mami Murakami, Elijus Undzys, Shyh-Dar Li. Stromal depletion by a docetaxel nanoparticle for enhanced therapy of breast and pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4486. doi:10.1158/1538-7445.AM2014-4486
Abstract 4488: Multi-functional, miR-205 complexed, gemcitabine conjugated nanomedicines for effective treatment of chemo-resistant pancreatic adenocarcinoma
Authors: Chitkara Deepak , Mittal Anupama , Behrman Stephen W. , Mahato Ram I.
Published: May 2015

Aberrant expression of small, non-coding RNAs called miRNAs in tumor tissues result in anomalous levels of genes and proteins which trigger metastasis and chemoresistance, and aggravate stemness among the cancer cells. We investigated one such miRNA, miR-205 identified through various softwares (TARGETSCAN, miRDB and DIANACOT) and validated experimentally as a potential target for treatment of advanced stage pancreatic cancer. Most of the current attempts to deliver such potent biologically complex molecules fail clinically owing to the challenges of structural and functional stability involved in their formulation and in vivo delivery. In this study, we constructed a multifunctional, self-assembling, copolymer with carboxyl pendant groups (mPEG-PCC) which exhibited efficient conjugation with gemcitabine (12.1% w/w) and simultaneously formed stable complexes with miR-205 at N/P 16/1. Both the therapeutic molecules which are otherwise known for plasma degradation demonstrated long-term stability in plasma when delivered through our system. Nanomedicines prepared using this polymer by film hydration method showed a size of ∼72.9 nm and sustained gemcitabine release of ∼40% in 10 days which was not affected by the co-presence of miRNA in the formulation. In gemcitabine resistant MIA PaCa-2R pancreatic cancer cells, the formulations showed a transfection efficiency of more than 90% (by flow cytometry), reversal of chemo-resistance (cell viability assay) and decrease in invasive and migratory ability of the cancer cells (Matrigel® invasion assays). In vivo evaluation in xenograft model developed using MIA PaCa-2R cells indicated significantly diminished tumor growth in miR-205/gemcitabine nanomedicines treated group.

Note: This abstract was not presented at the meeting.

Citation Format: Deepak Chitkara, Anupama Mittal, Stephen W. Behrman, Ram I. Mahato. Multi-functional, miR-205 complexed, gemcitabine conjugated nanomedicines for effective treatment of chemo-resistant pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4488. doi:10.1158/1538-7445.AM2014-4488
Abstract 4503: A small molecule glycomimetic antagonist of E-selectin (GMI-1271) prevents pancreatic tumor metastasis and offers a novel treatment for improved efficacy of chemotherapy
Authors: Steele Maria M. , Radhakrishnan Prakash , Magnani John L. , Hollingsworth Michael A.
Published: May 2015

The processes of intra- and extravasation of tumor cells into and out of the blood and lymphatic systems are crucial steps during metastasis to distant organ sites. These processes are tightly regulated by the initial binding of sialyl Lewis A and sialyl Lewis X (sialyl Le A/X) carbohydrate moeities found on tumor cells to the adhesion protein E-selectin expressed on the activated endothelium. GMI-1271 is a small molecule glycomimetic rationally designed based on the bioactive conformation of sialyl Lea/x and is a potent and specific antagonist of E-selectin. In vitro treatment of human lymphatic endothelial cells with GMI-1271 resulted in a decrease in the number of sialyl Lewis A-expressing pancreatic cancer cells (S2.013 and BxPC-3) binding to the endothelium in a dose-dependent manner. GMI-1271 treatment also inhibited the transendothelial migration of S2.013 and BxPC-3 cells through a lymphatic cell monolayer.

We evaluated the in vivo efficacy of GMI-1271 following orthotopic implantation of pancreatic tumor cell line S2.013, which expresses high levels of sialyl Lewis A (CA19-9), into nude mice. Following 2 weeks of tumor growth, mice were treated by intraperitoneal injections for 4 weeks with either PBS once daily, once daily with 40mg/kg GMI-1271 (low dose), twice daily with 40mg/kg GMI-1271 (high dose), twice a week with 60mg/kg gemcitabine injections, combination low dose GMI-1271 and gemcitabine injections, or combination high dose GMI-1271 and gemcitabine injections. Co-treatment of either low or high dose GMI-1271 with gemcitabine resulted in a significant decrease in the number of metastasis to the lymph nodes (p=0.02 low dose; p=0.04 high dose). In addition, compared with gemcitabine alone, low dose GMI-1271 plus gemcitabine was found to be effective at reducing the number of metastatic lesions (per histological section) in the liver (p=0.001), lung (p=0.026) and diaphragm (p=0.01). Based on the significant effects of combination therapy on tumor metastasis, the small molecule glycomimetic antagonist to E-selectin, GMI-1271, offers great promise in preventing pancreatic tumor cell entry into the blood and lymphatic systems and offers a novel treatment for the improved efficacy of standard chemotherapy.

Citation Format: Maria M. Steele, Prakash Radhakrishnan, John L. Magnani, Michael A. Hollingsworth. A small molecule glycomimetic antagonist of E-selectin (GMI-1271) prevents pancreatic tumor metastasis and offers a novel treatment for improved efficacy of chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4503. doi:10.1158/1538-7445.AM2014-4503