bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1785: Distinct effect of aldehydes in anthracycline cytotoxicity in S. cerevisiae
Authors: Miles Jana Sharrae , Mazu Tryphon , Darling-Reed Selina , Flores-Rozas Hernan
Published: May 2015

Anthracyclines are common chemotherapeutic agents used in many cancers which are refractory to other therapeutic alternatives. In triple negative breast cancer which is commonly observed in African-American women (AA) and tend to be more aggressive, carry worse prognoses and are harder to manage because they lack the molecular targets required for therapies, anthracyclines are some of the few therapeutic alternatives. Although effective, resistance to this agent usually arises and serious side-effects like the development of cardiomyopathy limits their use. To increase our knowledge of the pathways that protect cells from anthracyclines we carried out a genome-wide genetic screening in the eukaryotic model organism S. cerevisiae to identify gene defects that generate cells hypersensitive to doxorubicin. A total of 71 deletion strains displayed varying levels of sensitivity to anthracyclines. Among these, mutants of HOM6 confer significant increased sensitivity to doxorubicin. This effect appears to be due to the accumulation of the threonine intermediate L-aspartate-semialdehyde and can be abolished by the inactivation of a prior step, involving HOM3, which prevents the accumulation of the intermediate. In order to further characterize the synergism of aldehydes and doxorubicin, we have tested the effect of different aldehydes on the cytotoxicity of cells exposed to anthracyclines.

Our data show that treatment with formaldehyde (2 mM) reduced the survival of the wild type strain to 78%. However, in the presence of doxorubicin, formaldehyde reduced the viability of the strain by between 31 to 39-fold (with 10 µM doxorubicin) relative to doxorubicin and formaldehyde alone and by ∼110-fold relative to doxorubicin at 150 µM concentration.

Co-treatment with formaldehyde and doxorubicin of anthracycline-resistant strains siz1 and msh2 also shows increased toxicity. However, the synergistic effect is not as pronounced in these strains compared to the wild type. Among the aldehydes tested, formaldehyde was most effective. Glutaraldehyde and acetaldehyde displayed distinct effect on strains siz1, and ald5. The potential use of a combination of aldehydes and cytotoxic drugs could potentially lead to applications intended to enhance anthracycline-based therapy. This possibility will be discussed, as well as the targeting of aldehydes in relevant human pathways.

Research supported by NIH/NCRR RCMI grant #RR03020 at Florida A&M University.

Citation Format: Jana Sharrae Miles, Tryphon Mazu, Selina Darling-Reed, Hernan Flores-Rozas. Distinct effect of aldehydes in anthracycline cytotoxicity in S. cerevisiae. [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 1785. doi:10.1158/1538-7445.AM2014-1785
Abstract 1795: Small molecule SBI-601 inhibits Siah1/2 ubiquitin ligases, attenuates HIF1α expression and growth of melanoma and prostate cancer cells
Authors: Feng Yongmei , Varsano Tal , Sessions Hampton , Davies Michael , Roth Greg , Ronai Ze'ev
Published: May 2015

The ubiquitin ligases Siah1/2 have been implicated in the control of key cellular pathways, including hypoxia (through the control of prolyl hydroxylase 1/3 stability), mitochondrial fission (through the control of AKAP121 stability) and Ras/Raf signaling (through its control of Sprouty 2 stability). Expression or activity of Siah1/2 is upregulated in number of cancer types and correspondingly, inhibition of Siah1/2 effectively attenuates growth of prostate, melanoma, lung and pancreatic tumors. To identify possibly Siah1/2 inhibitors, we have performed a HTS screen using affinity selection-mass spectrometry (AS-MS). Using a select set of 32,000 drug-like molecules in mass-encoded mixtures of 400 compounds per assay well, we identified 36 distinct clusters of structurally related scaffolds that demonstrate high-binding affinity to full-length bacterially produced purified and active Siah2. Of those, 9 out of 21 closely related analogs were evaluated in functional assays. One compound, SBI-0640601, showed potent and reproducible inhibition of Siah2, as measured by the reduction in HIF1α levels and activity, measured by HRE-Luciferase assays in both melanoma and prostate cancer cultures. SBI-0640601 is a natural product known as betulonic acid (BA) and is one of a family of triterpenoid natural medicinal products derived from birch trees (genera Betula). Independent binding studies (differential scanning calorimetry) confirmed that SBI-0640601 binds to full-length bacterially produced Siah2 and to a RING domain-deleted version of Siah1. In vitro, SBI-0640601 attenuated the binding of PHD3 to Siah2, in keeping with the observation that ubiquitination of most substrates requires binding to the Siah2 substrate-binding domain. SBI-0640601 inhibited growth of melanoma and prostate cancer cells in 2D and 3D cultures. Further, SBI-0640601 effectively attenuated neuroendocrine differentiation phenotype in adenocarcinoma cells, consistent with the effect seen upon inhibition of Siah1/2 expression. RPPA analysis identified that SBI-0640601 effectively attenuates AMPK and mTOR signaling pathways, in addition to affecting MAPK signaling. Overall, SBI-0640601 appears to inhibit key processes previously shown to be regulated by Siah1/2, holding promise for further assessment in preclinical setting.

Note: This abstract was not presented at the meeting.

Citation Format: Yongmei Feng, Tal Varsano, Hampton Sessions, Michael Davies, Greg Roth, Ze'ev Ronai. Small molecule SBI-601 inhibits Siah1/2 ubiquitin ligases, attenuates HIF1α expression and growth of melanoma and prostate 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 1795. doi:10.1158/1538-7445.AM2014-1795
Abstract 1796: Novel triazole nucleoside inhibits heat shock factor 1 and androgen receptor to fight against prostate cancer
Authors: Xia Yi , Beraldi Eliana , Zoubeidi Amina , Peng Ling , Gleave Martin
Published: May 2015

Prostate cancer is one of the most commonly diagnosed cancers and leading cause of cancer-related mortality in men in developed countries. While most patients initially respond to first-line androgen deprivation therapy (ADT), many become refractory to hormonal and other conventional therapies and eventually develop castration-resistant prostate cancer (CRPC) with time. CRPC is often characterized by the reactivation of androgen receptor (AR) and constitutes the most important therapeutic target in CRPC.

It is known that AR is bound to heat shock protein (HSPs) complex to maintain its stable and mature conformation with high affinity to androgen. Targeting HSPs are therefore to result in perturbation of AR. Using a lead optimization process, we have recently identified a novel triazole nucleoside VM-1 which can down-regulate heat shock transcription factor 1 (HSF1), the dominant upstream transcriptional regulator for HSPs. The down-regulation of HSF1 by VM-1 leads to the simultaneous suppression of multiple HSPs and results in growth inhibition of various prostate cancer cells. Interestingly, VM-1 displays more potent antiproliferation activity in AR-positive LNCaP, C4-2 and 22RV-1 cells than AR-negative PC-3 cells. Our further studies have showed that VM-1 reduces the expression of AR and a panel of AR targeted genes as well as inhibits AR transactivation, which consequently leads to effective caspase-dependent apoptosis in LNCaP, C4-2 and 22RV-1 cells. Moreover, VM-1 arrests cell cycle of prostate cancer cells in AR-governed phase suggesting the potential functionality of this compound involved in the crosstalk between AR and cell cycle pathways. Taken together, the above data suggest the role of HSF1 in regulation of AR, and the inhibition on both HSF1 and AR contribute to the activity of VM-1 in prostate cancer cells. Therefore, VM-1 may serve as a novel structural motif in the search of anticancer candidates to fight against prostate cancer via inhibiting HSF1 and AR.

Citation Format: Yi Xia, Eliana Beraldi, Amina Zoubeidi, Ling Peng, Martin Gleave. Novel triazole nucleoside inhibits heat shock factor 1 and androgen receptor to fight against prostate 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 1796. doi:10.1158/1538-7445.AM2014-1796
Abstract 1797: Discovery of NVP-CGM097, a highly potent and optimized small molecule inhibitor of Mdm2 under evaluation in a Phase I clinical trial
Authors: Jeay Sebastien , Berghausen Joerg , Buschmann Nicole , Chène Patrick , Cozens Robert , Erdmann Dirk , Ferretti Stéphane , Furet Pascal , Gabriel Tobias , Gessier François , Graus-Porta Diana , Hofmann Francesco , Holzer Philipp , Ito Moriko , Jacoby Edgar , Jensen Michael , Kallen Joerg , Lang Marc , Lisztwan Joanna , Murakami Masato , Pissot-Soldermann Carole , Ruetz Stephan , Rynn Caroline , Sterker Dario , Stutz Stefan , Valat Thérèse , Wiesmann Marion , Masuya Keiichi
Published: May 2015

Activation of p53 by blocking the p53-Mdm2 interaction using non-peptidic small-molecule inhibitors is being pursued as a promising cancer therapeutic strategy. In the present study, we show the identification of NVP-CGM097, a novel, highly optimized, and selective inhibitor of the p53-Mdm2 interaction. NVP-CGM097 binds to human Mdm2 protein with a Ki value of 1.3 nM, activates p53 in human cells and induces robust p53-dependent cell cycle arrest and apoptosis in human p53 wild-type tumor cells. Its activity and selectivity has been tested and confirmed across a large panel of cancer cell lines from the Cancer Cell Line Encyclopedia. Importantly, NVP-CGM097 displays desirable pharmacokinetic and pharmacodynamic profiles in animals together with excellent oral bioavailability, which triggers rapid and sustained activation of p53-dependent pharmacodynamic biomarkers resulting in tumor regression in multiple xenografted models of p53 wild-type human cancer. The validation and understanding of its mechanism of action, the overall favorable drug-like properties and the characterization of its on-target toxicological profile in preclinical species strongly supported the initiation of Phase I clinical trials with NVP-CGM097 in pre-selected patients with p53 wild-type tumors.

Citation Format: Sebastien Jeay, Joerg Berghausen, Nicole Buschmann, Patrick Chène, Robert Cozens, Dirk Erdmann, Stéphane Ferretti, Pascal Furet, Tobias Gabriel, François Gessier, Diana Graus-Porta, Francesco Hofmann, Philipp Holzer, Moriko Ito, Edgar Jacoby, Michael Jensen, Joerg Kallen, Marc Lang, Joanna Lisztwan, Masato Murakami, Carole Pissot-Soldermann, Stephan Ruetz, Caroline Rynn, Dario Sterker, Stefan Stutz, Thérèse Valat, Marion Wiesmann, Keiichi Masuya. Discovery of NVP-CGM097, a highly potent and optimized small molecule inhibitor of Mdm2 under evaluation in a Phase I clinical trial. [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 1797. doi:10.1158/1538-7445.AM2014-1797
Abstract 180: Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces expression of fibronectin in human colon cancer cells
Authors: Shinagawa Kei , Kitadai Yasuhiko , Tanaka Yuichiro , Yuge Ryo , Onoyama Mieko , Tanaka Shinji , Yasui Wataru , Chayama Kazuaki
Published: May 2015

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to contribute to the formation of tumor stroma. We reported recently that in an orthotopic nude mouse model of human colon cancer, MSCs migrated to tumor stroma, where they differentiated into carcinoma-associated fibroblast (CAF)-like cells and promoted tumor growth and metastasis. However, the molecular mechanisms of this phenomenon have not been elucidated. We performed microarray analysis of gene expression in KM12SM cells and found that expression of fibronectin (FN) mRNA was increased markedly by direct co-culture of KM12SM cells with MSCs. FN, a glycoprotein in extracellular matrix and also a maker of epithelial-mesenchymal transition (EMT), has been implicated in the development of several types of human cancer. Many reports indicate that EMT is an important step during the cascade of tumor progression. However, the potential role of FN in carcinogenesis is not fully elucidated. We examined FN expression in surgical specimens of human colon cancer and colon adenoma by immunohistochemistry. A total of 87 tumors were analyzed. Levels of FN expression in tumor cells correlated with disease stage, and FN immunoreactivity was detected in invasive cancers, especially at the invasive edge, but less in mucosal cancers or adenomas. Furthermore, in an orthotopic nude mouse model of colon cancer, mRNA expression levels of FN were shown to be higher in the tumors produced by KM12SM cells mixed with MSCs than in the tumors produced by KM12SM cells alone.These findings suggest that the interaction between cancer cells and MSCs recruited into tumor stroma may play a role in tumor growth and metastasis via induction of FN expression in invasive colon cancer.

Citation Format: Kei Shinagawa, Yasuhiko Kitadai, Yuichiro Tanaka, Ryo Yuge, Mieko Onoyama, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Interaction between bone marrow-derived mesenchymal stem cells and tumor cells induces expression of fibronectin in human colon 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 180. doi:10.1158/1538-7445.AM2014-180
Abstract 1811: Evaluation of anti-CXCR2 small molecule inhibitors as novel chemotherapy targeting the Interleukin-8 pathway in colorectal cancer
Authors: Huang Yinghui Jane E. , Gaffney Kevin J. , Gerdts Ethan , Petasis Nicos A. , Lenz Heinz-Josef , LaBonte Melissa J.
Published: May 2015

Despite new and improved chemotherapy treatments for colorectal cancer (CRC) contributing to enhanced treatment response rates and patient prognosis, it has been suggested that an overwhelming 90% of metastatic CRC patients encounter treatment failure due to drug resistance. The Interleukin-8/CXCR2 pathway has been validated as a promising target for novel chemotherapy due to its overarching effects on various cell types within the tumor microenvironment that ultimately drive tumor growth, proliferation, survival, chemoresistance, metastasis and angiogenesis. A series of homologous small molecule inhibitors, which competitively bind to the active site of CXCR2, was developed and evaluated in terms of their efficacy in inhibiting the various cellular responses induced by IL-8/CXCR2 signaling, utilizing MTS cell proliferation, angiogenesis tube formation, colony formation, Western blot, cell migration and invasion assays. The anti-CXCR2 compounds developed produce potent repression of proliferation and colony forming capacity in HCT116, HT29, SW620 and LoVo CRC cell lines, achieving IC50 values as low as 9.6 μM in HCT116 and 9.6 μM in HT29, along with colony formation fold changes as low as .163 in HCT116 and .255 in HT29. Additionally, the compounds demonstrated inhibition of angiogenesis, migration and invasion and induced significant down-regulation of pathways downstream of CXCR2, including NF-κB, MAPK and Akt. The proposed targeting of the IL-8 pathway offers an attractive and promising approach to CRC therapy. Based on the structure and functional relationship of these compounds, new inhibitors will be designed and validated in hopes of having improved drug properties by utilizing our in vitro and in vivo assay systems.

Citation Format: Yinghui Jane E. Huang, Kevin J. Gaffney, Ethan Gerdts, Nicos A. Petasis, Heinz-Josef Lenz, Melissa J. LaBonte. Evaluation of anti-CXCR2 small molecule inhibitors as novel chemotherapy targeting the Interleukin-8 pathway in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1811. doi:10.1158/1538-7445.AM2014-1811
Abstract 1818: ERBB3/HER2 signaling limits the effectiveness of EGFR blockade in head and neck and colorectal cancer models
Authors: Zhang Li , Castanaro Carla , Luan Bo , Yang Katie , Fan Amy , Rudge John , Papadopoulos Nicholas , Thurston Gavin , Daly Christopher
Published: May 2015

Introduction: EGFR blocking antibodies are approved for the treatment of colorectal cancer (CRC) and head and neck squamous cell carcinoma (HNSCC). However, responses to single agent EGFR blocking antibodies are relatively infrequent and transient. Thus, there is a clear need for agents that can enhance the benefit of EGFR blockade. While ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. In this report, we employ blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955), as well as a HER2 blocking antibody, to investigate the role of ERBB3 in modulating the response to EGFR inhibition.

Experimental procedures and results: We show that EGFR and ERBB3 are co-activated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits the growth of these cell lines more effectively than blockade of either receptor alone. Western blotting analysis shows that REGN955 strongly inhibits activation of ERK, but not Akt, while REGN1400 strongly inhibits activation of Akt, but not ERK, consistent with the more potent effect of the combination on cell growth. In addition, using a colorectal cancer cell line with low endogenous ERBB3 activity, we show that neuregulin 1 treatment significantly increases ERBB3 phosphorylation and reverses the inhibitory effect of REGN955 on cell growth. To further understand the mechanism of ERBB3 activation, we employed a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and CRC cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on CRC cell growth. Finally, while REGN1400 and REGN955 as single agents slowed the growth of HNSCC and CRC xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression.

Summary and conclusion: Our findings demonstrate that ERBB3 partners with HER2 to activate the Akt survival pathway and to limit the effect of EGFR blockade on the growth of HNSCC and CRC cell lines and tumor xenografts. These findings suggest that REGN1400, which is currently in a Phase 1 clinical trial, could provide clinical benefit when combined with EGFR blocking antibodies.

Citation Format: Li Zhang, Carla Castanaro, Bo Luan, Katie Yang, Amy Fan, John Rudge, Nicholas Papadopoulos, Gavin Thurston, Christopher Daly. ERBB3/HER2 signaling limits the effectiveness of EGFR blockade in head and neck and colorectal cancer models. [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 1818. doi:10.1158/1538-7445.AM2014-1818
Abstract 1839: LKB1 deficiency enhances sensitivity to energetic stress induced by erlotinib treatment in non-small cell lung cancer (NSCLC) cells
Authors: Whang Young M. , Park Serk I. , Trenary Irina A. , Lee Changki , Kaufman Jacob M. , Carbone David P. , Young Jamey D.
Published: May 2015

The tumor suppressor LKB1 is inactivated in 20-30% of non-small cell lung cancer (NSCLC) patient tumors. Because LKB1 is the master kinase phosphorylating the energy-sensing kinase AMPK, dysregulation of the LKB1-AMPK signaling axis causes loss of cellular homeostasis in response to metabolic inhibition or external stress-induced mitochondrial insults. Erlotinib is used clinically in NSCLC patients to target mutationally activated EGFR and is known to induce mitochondrial-mediated apoptosis. We investigated the metabolic response and mitochondrial function of LKB1-deficient NSCLC cells treated with erlotinib. LKB1-deficient cells exhibited enhanced sensitivity to erlotinib treatment in vitro and in vivo despite having wild-type EGFR. We found that this enhanced response was due to altered energetic metabolism and mitochondrial dysfunction in LKB1-deficient cells, which prevents homestatic maintenance of ATP and reactive oxygen species (ROS) levels in response to erlotinib treatment. Despite negative regulation of mTOR activity by LKB1-AMPK signaling, erlotinib effectively blocked mTOR signaling in LKB1-deficient cells, resulting in inhibition of cell growth, as well as activation of apoptosis. These findings will be important for designing targeted treatments for LKB1-deficient NSCLC patient tumors harboring wild-type EGFR, which disrupt metabolic and signaling pathways known to regulate energy metabolism in these cells.

Citation Format: Young Mi Whang, Serk In Park, Irina A. Trenary, Changki Lee, Jacob M. Kaufman, David P. Carbone, Jamey D. Young. LKB1 deficiency enhances sensitivity to energetic stress induced by erlotinib treatment in non-small cell lung cancer (NSCLC) 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 1839. doi:10.1158/1538-7445.AM2014-1839
Abstract 1843: Primary and acquired resistance to HERs inhibition in colorectal cancer cells: All HERs go to RAS
Authors: Russo Mariangela , Riggio Alessandra , Cancelliere Carlotta , Nicolantonio Federica D. , Bardelli Alberto
Published: May 2015

The monoclonal antibodies cetuximab and panitumumab are approved for the treatment of metastatic colorectal cancers (mCRC). When used as monotherapy, the anti-EGFR antibodies are effective in approximately 15% of patients. It is now well established that colorectal tumors carrying KRAS and BRAF mutations (40%) are refractory to EGFR targeted therapies (primary resistance). Virtually all-responsive patients eventually become insensitive to anti-EGFR antibodies (secondary resistance) within 3 to 12 months of initiating therapy. Primary and acquired resistance therefore limits the clinical benefit of EGFR blockade in CRC patients.

We postulated that concomitant inhibition of multiple members of the HER (ErbB) receptors family might suppress the growth of CRC cells more effectively than selectively targeting only the EGFR with cetuximab or panitumumab.

Accordingly, we assessed the pharmacological activity of momelotinib and afatinib- two pan-HER tyrosine kinase inhibitors- in a collection of 40 CRC cell lines, which recapitulate the genetic profiles commonly detected in CRC patients. We report that primary resistance to pan-HER inhibition is associated with KRAS and BRAF mutations thus suggesting that this patients' population will unlikely benefit from momelotinib and afatinib treatments. Notably, however, pan-HER blockade was effective in a subset of KRAS/BRAF wild type CRC cell lines refractory to cetuximab and panitumumab indicating that a subgroup of patients, which do not benefit from EGFR blockade, may profit from pan-HER inhibition.

To define the molecular mechanisms of acquired resistance to ErbB inhibitors, three KRAS/BRAF/NRAS wild type CRC cell models were continuously treated with cetuximab, momelotinib and afatinib, until resistant derivatives emerged. Genetic characterization of such derivatives unveiled that, regardless of the drug used, the resistant populations were a mixture of clones carrying distinctive mutations in KRAS or NRAS. Detailed biochemical analyses of the ErbB signalling pathway revealed that resistant variants displayed high levels of MEK and ERK activation, which persisted upon ErbBs inhibition.

Collectively, these results identify KRAS, BRAF and NRAS mutations as major drivers of primary and/or acquired resistance to HERs inhibition in colorectal cancer cells and suggest that a molecularly defined subset of CRC patients might benefit from pan-HER blockade.

Citation Format: Mariangela Russo, Alessandra Riggio, Carlotta Cancelliere, Federica Di Nicolantonio, Alberto Bardelli. Primary and acquired resistance to HERs inhibition in colorectal cancer cells: All HERs go to RAS. [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 1843. doi:10.1158/1538-7445.AM2014-1843
Abstract 1849: High nuclear expression of the p53 target Wig-1 is associated with poor prognosis in cervical carcinoma
Authors: Xu Lidi , Muller Susanne , Lerner Mikael , Lagercrantz Svetlana , Grandér Dan , Wallin Keng-Ling , Wiman Klas G. , Andersson Sonia , Larsson Catharina
Published: May 2015

Cervical cancer is the third most common cancer in women worldwide, and is the most common cancer in Eastern Africa, South-Central Asia and Melanesia. Persistent HPV infection is an important feature of cervical cancer. The central role of chromosome 3q for progression from low-grade dysplastic lesions to higher grades and to invasive carcinomas has been well established. Gain of 3q can occur in morphologically normal cytology of women who developed cervical carcinomas after only a short latency. The Wig-1 gene (wt p53-induced gene 1) is located at the frequently gained chromosomal region 3q. We examined Wig-1 in eight cervical cancer cell lines (Ca Ski, ME-180, MS751, SiHa, SW 756, C-4I, C-33A, and HT-3) by FISH, Southern, Northern, qRT-PCR, and Western blotting. We did not detect any Wig-1 gene alterations in these cell lines. C33-A and HT-3 with strong Wig-1 mRNA expression were HPV negative while the HPV positive cells only exhibited low / moderate Wig-1 expression by Northern blotting. However, no association was found between Wig-1 protein expression and HPV infection. We also assessed Wig-1 expression in a series of 38 cervical tumors, including both squamous cell carcinoma (SCC) and adenocarcinoma (ADCA), by immunohistochemistry. Interestingly, patients who showed moderate nuclear Wig-1 expression levels and positive cytoplasmic Wig-1 staining in their tumors had significantly better prognosis than patients with high nuclear Wig-1 expression and no cytoplasmic staining (Log Rank P value = 0.042). Moreover, higher nuclear Wig-1 expression levels were associated with HPV negative cervical tumors (P=0.003). These results suggest that a marker like Wig-1 could have considerable impact on prediction of the prognosis in women with cervical cancer. Our previous results indicate that Wig-1 acts as a survival factor. Thus, high levels of nuclear Wig-1 may promote tumor growth and survival. The exact role of Wig-1 at the molecular level during cervical carcinoma development needs further investigation.

Citation Format: Lidi Xu, Susanne Muller, Mikael Lerner, Svetlana Lagercrantz, Dan Grandér, Keng-Ling Wallin, Klas G. Wiman, Sonia Andersson, Catharina Larsson. High nuclear expression of the p53 target Wig-1 is associated with poor prognosis in cervical 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 1849. doi:10.1158/1538-7445.AM2014-1849
Abstract 1855: SCT methylation is a potential cancer biomarker for lung cancer
Authors: Zhang Yu-An , Ma Xiaotu , Fujimoto Junya , Wistuba Ignacio , Lam Stephen , Stastny Victor , Gao Boning , Larsen Jill , Liu Xiaoyun , Minna John D. , Zhang Michael Q. , Gazdar Adi F.
Published: May 2015

Purpose: To validate SCT (secretin) methylation as a potential cancer biomarker for lung cancer. Background: There is an urgent need for specific and sensitive biomarkers for lung cancer. The human secretin (SCT) gene is located on chromosome 11p15.5 and encodes the precursor gene of secretin, an endocrine hormone. From analysis of The Cancer Genome Atlas methylation array data, we (XM and MQZ) observed that the exon 1 promoter region of SCT gene is differentially hypermethylated in lung cancer (unpublished). These observations led us to further validate whether SCT methylation can be a useful cancer biomarker for lung cancer. Methods/Results: (1). We designed a novel nested PCR assay for SCT bisulfite sequencing of the promoter and exon 1 region of SCT. Bisulfite sequencing showed that SCT was fully methylated in nearly all CpG sites examined in 10 lung cancer cell lines, whereas SCT was non-methylated in 11 normal WBC blood cells. Accordingly we designed a semi-quantitative SCT qMSP assay. (2). We applied SCT qMSP on genomic DNA from frozen tissues of 131 lung tumors and 65 non-malignant adjacent lung tissues (53 paired). The level of SCT methylation was significantly higher (>24 fold) in all 111 malignant lung tumors (64 adenocarcinomas, 19 squamous cell carcinomas, 12 large cell neuroendocrine carcinomas, 16 NSCLC-other type), as compared to 65 non-malignant adjacent lung tissues. The high SCT methylation was further confirmed in malignant lung tumor formalin-fixed paraffin-embedded (FFPE) tissues samples (8 pairs tested). SCT methylation distinguishes malignant lung from non-malignant adjacent lung with area under curve (AUC) score of 0.92 (p<0.0001). (3). Interestingly, SCT hypermethylation does not significantly distinguishes low grade lung carcinoids tumors (n=20) from non-malignant adjacent lung with AUC score of 0.54 (p=0.5759). (4). Nearly full SCT methylation was detected in lung cancer cell lines (n=27) as compared to absent or lower levels of SCT methylation in immortalized human respiratory cell lines (n=38). Conclusions: SCT methylation is a highly specific and sensitive tissue biomarker for malignant lung cancer and can be detected in FFPE samples. SCT methylation is infrequent in low grade lung carcinoids tumors. Its clinical uses need to be explored.

Citation Format: Yu-An Zhang, Xiaotu Ma, Junya Fujimoto, Ignacio Wistuba, Stephen Lam, Victor Stastny, Boning Gao, Jill Larsen, Xiaoyun Liu, John D. Minna, Michael Q. Zhang, Adi F. Gazdar. SCT methylation is a potential cancer biomarker for 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 1855. doi:10.1158/1538-7445.AM2014-1855
Abstract 1868: Clinical significance of cathepsin L and B expression in pediatric acute myeloid leukemia
Authors: Pandey Garima , Bakhshi Sameer , Singh Ratnakar , Chauhan Shyam S.
Published: May 2015

Introduction: Cathepsin L and B are commonly expressed cysteine proteinases that play a major role in lysosomal bulk proteolysis, protein processing, matrix degradation, and tissue remodelling. In tumors there is deregulation of these proteases that interferes with these normal biological processes. In a pilot study done in our lab expression of cathepsin L and B were found to be significantly elevated in peripheral blood mononuclear cells (PBMCs) of pediatric AML patients. However the expression of these proteases in bone marrow aspirate, which represents almost true population of blasts, has never been studied before.

Methods: Enzymatic activities of cathepsin L and B were assayed in PBMCs and bone marrow mononuclear cells (BMMCs) of AML patients and healthy controls. Further expression of these proteases and its inhibitor were confirmed by western blotting and real time assay. Cell cycle analysis in PBMCs and BMMCs of these patients was performed by flow cytometry using propidium iodide.

Results: In the PBMCs of pediatric AML patients activity of cathepsin L and B was found to be significantly elevated as compared to healthy controls. Similarly, increased activity of cathepsin L and B was also observed in the BMMCs when compared with PBMCs of these patients. Furthermore, protein and mRNA expression also confirmed the increased level of these proteases in pediatric AML. The protein and mRNA expression of their endogenous inhibitor cystatin-C was found to be decreased in PBMCs and BMMCs of AML patients. Moreover, expression of these proteases correlated positively with the percentage of blasts and percentage of cells in S-phase.

Conclusion: This study for the first time demonstrates the increased expression of cathepsin L and B in the BMMCs and PBMCs of pediatric AML patients, along with a positive correlation with percentage of blasts, thereby suggesting a potential role of these proteases in the severity of this disease.

Citation Format: Garima Pandey, Sameer Bakhshi, Ratnakar Singh, Shyam S. Chauhan. Clinical significance of cathepsin L and B expression in pediatric acute myeloid leukemia. [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 1868. doi:10.1158/1538-7445.AM2014-1868
Abstract 1870: Development of a standard operating procedure for exosome isolation and analysis using clinical samples: Application to cancer biomarker discovery
Authors: Setterquist Robert A. , Rai Alex J. , Zeringer Emily , Li Mu , Barta Tim , Schageman Jeoffrey , Magdaleno Susan , Vlassov Alexander V.
Published: May 2015

Exosomes are small vesicles (30-150 nm) found in abundance in human body fluids which function as carriers of different species of RNA and protein between diverse locations in the body. The spectrum of current scientific interest in exosomes is wide and ranges from studying their functions and pathways to utilizing them in diagnostics and therapeutics development. As such, there is a growing need for quick and easy methods for both isolation of exosomes and analysis of their cargo.

We present herein a workflow for exosome isolation and analysis which entails: (i) fast and efficient isolation of exosomes from serum, plasma, and urine of both healthy donors and patients with prostate cancer, using Total Exosome Isolation reagents; (ii) characterization of their size distribution and count with Nanosight LM10 instrument; (iii) extraction of exosome “cargo” with Total Exosome RNA and Protein Isolation kit; (iv) characterization of exosomal RNA content using the Ion Torrent PGM sequencing and qRT-PCR.

The protocol described herein lays the groundwork for the development of a standardized operating procedure (SOP) for isolation of exosomes and downstream analysis of their constituents, using clinical samples.

We demonstrate that cancer-specific RNA signatures residing within the exosomes can be delineated from different patient cohorts. This is the first step towards developing a method whereby performance characteristics can be measured and used to optimize a validated assay useful for routine testing of clinical samples.

Citation Format: Robert A. Setterquist, Alex J. Rai, Emily Zeringer, Mu Li, Tim Barta, Jeoffrey Schageman, Susan Magdaleno, Alexander V. Vlassov. Development of a standard operating procedure for exosome isolation and analysis using clinical samples: Application to cancer biomarker discovery. [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 1870. doi:10.1158/1538-7445.AM2014-1870
Abstract 1905: Defining a therapeutic classification of breast cancer by actionable targets
Authors: Salto-Tellez Manuel , Boyle David P. , McArt Darragh , Irwin Gareth , Wilhelm-Benartzi Charlotte Charlotte , LIoe Tong G. , Minter Martha , McQuaid Stephen , Mullan Paul , Kennedy Richard D. , Hamilton Peter , Harkin D P.
Published: May 2015

Breast cancer remains a frequent cause of female cancer death despite the great strides in elucidation of biological subtypes and their reported clinical and prognostic significance. We have defined a general cohort of breast cancers in terms of putative actionable targets, involving growth and proliferative factors, the cell cycle, and apoptotic pathways, both as single biomarkers across a general cohort and within intrinsic molecular subtypes.

We identified 293 patients treated with adjuvant chemotherapy. Additional hormonal therapy and trastuzumab was administered depending on hormonal and HER2 status respectively. We performed immunohistochemistry for ER, PR, HER2, MM1, CK5/6, p53, TOP2A, EGFR, IGF1R, PTEN, p-mTOR and e-cadherin. The cohort was classified into luminal (62%) and non-luminal (38%) tumors as well as luminal A (27%), luminal B HER2 negative (22%) and positive (12%), HER2 enriched (14%) and triple negative (25%). Patients with luminal tumors and co-overexpression of TOP2A or IGF1R loss displayed worse overall survival (p=0.0251 and p=0.0008 respectively). Non-luminal tumors had much greater heterogeneous expression profiles with no individual markers of prognostic significance. Non-luminal tumors were characterised by EGFR and TOP2A overexpression, IGF1R, PTEN and p-mTOR negativity and extreme p53 expression.

Our results indicate that only a minority of intrinsic subtype tumors purely express single novel actionable targets. This lack of pure biomarker expression is particular prevalent in the triple negative subgroup and may allude to the mechanism of targeted therapy inaction and myriad disappointing trial results. Utilising a combinatorial biomarker approach may enhance studies of targeted therapies providing additional information during design and patient selection while also helping decipher negative trial results.

Citation Format: Manuel Salto-Tellez, David P. Boyle, Darragh McArt, Gareth Irwin, Charlotte Charlotte Wilhelm-Benartzi, Tong G. LIoe, Martha Minter, Stephen McQuaid, Paul Mullan, Richard D. Kennedy, Peter Hamilton, D P. Harkin. Defining a therapeutic classification of breast cancer by actionable targets. [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 1905. doi:10.1158/1538-7445.AM2014-1905
Abstract 1908: The Wnt-target Prox1 promotes colorectal cancer stem cell survival to fuel tumor growth
Authors: Wiener Zoltan , Hyvönen Ville , Högström Jenny , Holopainen Tanja , Band Arja , Kallio Pauliina , Dufva Olli , Haglund Caj , Kruuna Olli , Oliver Guillermo , Ben-Neriah Yinon , Alitalo Kari
Published: May 2015

Colorectal cancer (CRC) is one of the leading causes of cancer mortality in the Western countries. In most of the CRC patients, an initial mutation occurs in the APC or CTNNB1 genes, leading to the ligand-independent activation of the canonical Wnt pathway. Besides having a central role in the development of CRC, the Wnt pathway plays a critical role also in the maintenance of the normal intestine. Attempts at therapeutic inhibition of this pathway could thus lead to serious side effects in CRC patients. We have previously shown that the Prox1 transcription factor is induced in the intestinal epithelium by mutations activating the Wnt pathway and it critically contributes to CRC progression via an unknown mechanism. Here we provide evidence that Prox1 expression is induced in the Lgr5+ adenoma stem cells early after Apc deletion. Our in vivo models and ex vivo organoid experiments suggest that Prox1 silencing or deletion restricts the expansion of the Lgr5+ adenoma stem cell population both in humans and in mice. Interestingly, silencing the phospholipid binding protein Annexin A1 (Anxa1), a gene suppressed by Prox1, is responsible for several of the effects of Prox1 on adenoma cells, such as the re-organization of the actin cytoskeleton, enhanced stem cell activity and tumor growth. Furthermore, Prox1 deletion abnormally increases the mTORC1 pathway activity, which results in decreased survival of the adenoma stem cells. Since Prox1 is expressed at low level only in rare neuroendocrine cells and in some Paneth cells in the wild type intestinal epithelium, furthermore, its genetic deletion in the adult gut does not lead to obvious phenotypes, Prox1 may serve as an attractive therapeutic target for restricting the progression of early intestinal adenomas.

Citation Format: Zoltan Wiener, Ville Hyvönen, Jenny Högström, Tanja Holopainen, Arja Band, Pauliina Kallio, Olli Dufva, Caj Haglund, Olli Kruuna, Guillermo Oliver, Yinon Ben-Neriah, Kari Alitalo. The Wnt-target Prox1 promotes colorectal cancer stem cell survival to fuel tumor growth. [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 1908. doi:10.1158/1538-7445.AM2014-1908
Abstract 191: Desmoplasia in primary tumors and metastatic lesions of pancreatic cancer
Authors: Whatcott Clifford J. , Watanabe Aprill , LoBello Janine , Hoff Daniel Von , Han Haiyong
Published: May 2015

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death in the United States. PDAC is characterized by high levels of fibrosis, a feature termed desmoplasia. Desmoplasia is thought to hamper the efficacy of therapeutics treating PDAC. Agents targeting components of the tumor stroma have shown some enhancement of first-line therapeutics targeting tumor cells. As metastatic burden is one of the primary causes for mortality in PDAC, the question remains of whether stromal targeting therapeutics will benefit patients with late stage, metastatic disease. We sought to address this question by assessing the extent of desmoplasia in both primary PDAC tumors and metastatic lesions of pancreatic origin. Patient tissue staining using a pentachrome or standard immunohistochemical technique demonstrates that extracellular matrix components, such as collagen, are found in high levels in both primary tumors and metastatic lesions. The difference in the level of desmoplasia in primary tumors and metastatic lesions was statistically insignificant. We also observed a significant correlation between patient survival and extracellular matrix deposition. Kaplan-meier curves for collagen I showed median survival of 527 days in low collagen patients, and 176.5 in high level patients. Low level hyaluronan patients displayed median survival times of 2107 days as compared to 385 days in high level patients. Our results indicate that both primary tumors and metastases of PDAC have highly fibrotic stroma. Thus, stromal targeting agents may benefit PDAC patients, even those with metastatic disease.

Citation Format: Clifford J. Whatcott, Aprill Watanabe, Janine LoBello, Daniel Von Hoff, Haiyong Han. Desmoplasia in primary tumors and metastatic lesions of 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 191. doi:10.1158/1538-7445.AM2014-191
Abstract 1911: NF-κB mediated CD47 upregulation promotes sorafenib resistance and its blockade synergizes the effect of sorafenib in hepatocellular carcinoma
Authors: Lo Jessica , Lau Eunice Yuen Ting , Ng Irene Oi Lin , Lee Terence Kin Wah
Published: May 2015

Sorafenib, a multikinase inhibitor, is currently used as the normative treatment for advanced hepatocellular carcinoma (HCC). It can prolong survival for a few months however sorafenib insensitivity and resistance often develops in tumors hence a better understanding of resistance mechanisms is urgently needed. Tumor-initiating cells (T-ICs) have recently been implicated in the cause of treatment resistance. Recently, our group found that CD47 is preferentially expressed in liver T-ICs, which suggests a possibility of targeting CD47 in order to evade sorafenib resistance via elimination of liver T-ICs. To test this hypothesis, we have successfully developed sorafenib-resistant clones in HCC cell lines (BEL7402 and Huh-7 cells) and in vivo using patient-derived xenograft (PDTX #1) by continuous exposure to sorafenib. We found that sorafenib-resistant clones showed enhanced T-IC properties such as self-renewal, tumorigenicity and invasiveness, which is also accompanied by an increase in CD47 expression. We found increased NF-κB activation in sorafenib resistant clones indicated by upregulated levels of phosphorylated p65 and IkBα which is consistent to the bioinformatics analysis showing two putative NF-κB binding sites on the CD47 promoter. In addition, CD47 expression was found to be decreased upon treatment of NF-κB inhibitor IMD-0354 and increased upon treatment of TNF-α. These results suggested that NF-κB mediated CD47 upregulation promotes sorafenib resistance in HCC. To further confirm the role of CD47 in sorafenib resistance, we knocked down CD47 expression in CD47-high expressing HCC cells. Through annexin V staining, we found that knockdown of CD47 sensitized Huh-7 and MHCC-97L to sorafenib treatment at 10µM and 20µM respectively. Using patient derived xenograft model (PDTX #8) to investigate the effect of different treatment regimens, we found that mice treated with daily administration of sorafenib (100mg/Kg) showed tumor volume reduction by 3-fold upon 30 days of treatment and a similar effect was found in mice treated daily with and anti-CD47 antibody (500mg/Kg). Interestingly, sorafenib combined with anti-CD47 antibody exhibited maximal effect on tumor suppression. All in all, regulation of CD47 by NF-κB may enable the promotion of sorafenib resistance and anti-CD47 antibody in co-treatment with sorafenib may serve as a novel therapeutic regimen for the treatment of advanced stage HCC.

Citation Format: Jessica Lo, Eunice Yuen Ting Lau, Irene Oi Lin Ng, Terence Kin Wah Lee. NF-κB mediated CD47 upregulation promotes sorafenib resistance and its blockade synergizes the effect of sorafenib in hepatocellular 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 1911. doi:10.1158/1538-7445.AM2014-1911