bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 269: Asymmetric laterality of breast tumors is correlated with place of birth, but not race
Authors: Sughue Trevor , Brody James P.
Published: May 2015

More breast cancers are diagnosed in the left breast than the right breast. The ratio of the number of tumors diagnosed in the left breast to the number diagnosed in the right in a population is called the laterality ratio. Previous studies showed that the breast cancer laterality ratio is significantly larger than 1 and significantly different in different quadrants of the breast.

We analyzed 1.2 million cases of breast cancer diagnosed in the US between 1973 and 2010 and recorded by the Surveillance, Epidemiology, and End Results (SEER) program data, November 2012 submission.

We found that the laterality ratio depends upon the country of birth, but not race of the patient. We identified five countries of birth that had p-values larger than 0.995, while we expected to see less than 1. Those born in Japan (l/r=1.14, p=0.997), the Ryuukyu Islands (l/r= 2.6, p=0.998), Laos (l/r=1.62, p=0.9999) and Algeria (l/r=2.1 p=0.9959) had significantly larger laterality ratios compared to the overall SEER population (l/r=1.04). Those born in Poland (l/r=0.92, p=0.997) had a laterality ratio significantly less than expected.

The SEER data classified patients into 30 different race categories. We expect about six of these categories to exceed the two tailed 90% confidence interval based upon chance. We found seven, three of which had p-values exceeding 0.95, but none of which exceeded 0.99. This finding suggests no correlation exists between race and laterality.

Next we sought to establish whether the difference in birthplace laterality ratios arose from (1) different distributions of the quadrant in which the tumor occurs or (2) a shift in the laterality ratio of each quadrant where the tumor occurs. First, we compared the distribution of quadrants in which a breast tumor occurs in those born in Poland with those born in Japan. We found that the relative distributions of tumor quadrants were identical. Next, we compared the laterality ratio by quadrant for the two populations. We found the only significant difference was in the laterality ratio of the upper outer quadrant. Thus, the birthplace effect appears to only occur in the upper outer quadrant of the breast.

Finally, we found a small, but statistically significant, increase in the breast cancer laterality ratio with age, and decrease with birth year and year of diagnosis.

Citation Format: Trevor Sughue, James P. Brody. Asymmetric laterality of breast tumors is correlated with place of birth, but not race. [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 269. doi:10.1158/1538-7445.AM2014-269
Abstract 2694: Novel sesquiterpene lactones derived from Vernonia cinerea inhibit STAT3 activity and suppress human glioblastoma phenotype
Authors: Miklossy Gabriella , Youn Ui Joung , Yue Peibin , Chang Leng Chee , Turkson James
Published: May 2015

Constitutively-active STAT3 is prevalent in glioblastoma multiforme, breast cancer and many other human tumors and is a valid target for the discovery of novel anticancer therapeutics. Screening of whole plant extracts from Vernonia cinerea identified sesquiterpene lactones of the hirsutinolide type that inhibit aberrant STAT3 activity and suppress human glioma phenotype in vitro and in vivo. Compounds 6, 10, 20 and 22 suppressed the viability of human glioma, U251MG cells that harbor aberrantly-active STAT3, with IC50 of 2.4-6 µM. Treatment of U251MG cells with any of the four natural product agents inhibited STAT3 phosphorylation in a time-dependent manner, with little or no effects on pErk1/2 and pAkt levels. By contrast, other hirsutinolides, including compounds 8, 9, 11, 18, 19 and 28 showed modest or undetectable effects on U251MG cell viability or on pSTAT3 in U251MG cells. Furthermore, treatment with compounds 6, 10 or 22 had moderate or no effects on phospho-JAK2 or phospho-Src or on the protein tyrosine phosphatase, PTP1B level, as measured by Western blot. Moreover, combined treatment with sodium orthovanadate had minimal effect on compound 6-induced decrease in pSTAT3 levels, altogether suggesting that protein tyrosine phosphatases are less likely to be the primary mechanism for down-regulation of pSTAT3 in treated U251MG cells. By contrast, co-treatment with compund 6 repressed the robust induction of pSTAT3 levels by sodium orthovanadate. Furthermore, treatment with compound 6, 10, 20 or 22 inhibited colony formation and the migration of U251MG cells in vitro, and repressed the expression of STAT3 regulated genes, including Mcl-1 in U251MG cells. In vivo anti-tumor efficacy studies of compound 6 revealed dose-dependent inhibition of the growth of human glioma tumor xenografts in mice. Our studies have identified natural product-based small molecules that potently inhibit STAT3 activity and preferentially induce antitumor cell effects in vitro and antitumor response in vivo against human glioma.

Citation Format: Gabriella Miklossy, Ui Joung Youn, Peibin Yue, Leng Chee Chang, James Turkson. Novel sesquiterpene lactones derived from Vernonia cinerea inhibit STAT3 activity and suppress human glioblastoma phenotype. [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 2694. doi:10.1158/1538-7445.AM2014-2694
Abstract 2759: Colorectal cancer cell lines with high BCL-XL and low MCL-1 expression are sensitive to a potent and selective BCL-XL inhibitor
Authors: Lam Lloyd T. , Zhang Haichao , Xue John , Hessler Paul , Tahir Stephen K. , Chen Jun , Jin Sha , Souers Andrew J. , Leverson Joel D.
Published: May 2015

Colorectal cancer is the second most deadly type of cancer in the United States and worldwide despite the recent approval of new therapies. The identification of novel agents that are effective against this cancer thus remains a significant goal. One hallmark of cancer is the defective regulation of programmed cell death, or apoptosis. The anti-apoptotic B-cell lymphoma 2 (BCL-2) family members, including BCL-2, BCL-XL, and MCL-1, are known to regulate cell survival and death and have been characterized as key survival factors in multiple cancer types. As BCL-XL is overexpressed in many solid tumors, the sensitivity of a panel of 25 colorectal cancer cell lines to a potent and selective BCL-XL inhibitor was investigated. Colorectal cell lines with high expression of BCL-XL and NOXA, a pro-apoptotic protein that antagonizes MCL-1 activity, were more sensitive to small molecule BCL-XL inhibition. In contrast, cell lines with lower expression of BCL-XL and higher expression of MCL-1 were resistant. Silencing the expression of BCL-XL via siRNA killed the cell lines that were sensitive to small molecule BCL-XL inhibition while having little effect on lines that were resistant. Furthermore, silencing the expression of MCL-1 restored sensitivity of resistant cells to the BCL-XL inhibitor whereas silencing of NOXA abrogated the sensitivity of cells to BCL-XL inhibitor. BCL-XL inhibition could thus be an effective treatment in a subset of colorectal cancer patients showing high BCL-XL expression.

Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for the research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Citation Format: Lloyd T. Lam, Haichao Zhang, John Xue, Paul Hessler, Stephen K. Tahir, Jun Chen, Sha Jin, Andrew J. Souers, Joel D. Leverson. Colorectal cancer cell lines with high BCL-XL and low MCL-1 expression are sensitive to a potent and selective BCL-XL inhibitor. [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 2759. doi:10.1158/1538-7445.AM2014-2759
Abstract 290: Constitutional BRCA1 methylation is a major predisposition factor for high-grade serous ovarian cancer
Authors: Dobrovic Alexander , Mikeska Thomas , Alsop Kathryn , Candiloro Ida , George Joshy , Mitchell Gillian , Bowtell David
Published: May 2015

This study aimed to determine whether constitutional methylation of the BRCA1 promoter region was a predisposition factor for ovarian cancer. We had previously shown that in a case control study of early-onset breast cancer patients, the presence of constitutional BRCA1 methylation in the blood was associated with tumors that phenocopied pathogenic germline BRCA1 mutations, indicating that BRCA1 methylation predisposed to and drove the development of these tumors. Germline BRCA1 mutations also predispose to ovarian cancer, in particular, high-grade serous ovarian cancer. In collaboration with the Australian Ovarian Cancer Study, we determined the presence of detectable BRCA1 methylation in 154 high-grade serous cancers. Twenty patients showed detectable levels of BRCA1 methylation in DNA extracted from their peripheral blood. Remarkably, when the corresponding tumor samples were assessed for methylation, 14 of the 20 that showed any methylation corresponded to a highly methylated tumour. When tumors occurring before the age of 55 were considered, 9 of the 10 bloods with detectable mutation corresponded to a strongly methylated tumour. This study also determined the frequency of BRCA1 and BRCA2 mutations. Fifteen women had germline BRCA1 mutations and 11 had germline BRCA2 mutations. The germline BRCA1 mutation carriers were predominantly younger at diagnosis and 11 pathogenic BRCA1 mutations were seen in the under 55 group. BRCA1 methylation was mutually exclusive with BRCA1 mutation. This data strongly suggests that constitutional BRCA1 methylation can drive ovarian cancer, in particular early onset ovarian cancer, and moreover is as important a predisposition factor as BRCA1 mutation.

Citation Format: Alexander Dobrovic, Thomas Mikeska, Kathryn Alsop, Ida Candiloro, Joshy George, Gillian Mitchell, David Bowtell. Constitutional BRCA1 methylation is a major predisposition factor for high-grade serous ovarian 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 290. doi:10.1158/1538-7445.AM2014-290
Abstract 2929: The Mdm2 inhibitor NVP-CGM097 enhances the anti-tumor activity of NVP-LDK378 in ALK mutant neuroblastoma models
Authors: Wang Hui Qin , Battalagine Linda , Liang Jinsheng , Halilovic Ensar , Schlegel Robert , Huang Alan , Cao Z. Alexander , Monahan John , Li Fang
Published: May 2015

Neuroblastoma is the most common cancer in infancy, accounting for 15% of all childhood cancer-related death. MYCN amplification is the major genetic aberration in high-risk neuroblastoma and is associated with poor outcome. Genome-wide association studies have identified activation mutations and high-level amplification of ALK in approximately 10% of neuroblastoma patients. In addition, ALK mutations can coexist with MYCN amplification, which defines a subset of ultra-high-risk neuroblastoma patients. In contrast to the high frequency of p53 mutations observed in many human cancers of adults, mutations of p53 are less common in childhood cancers and have been reported in less than 2% of neuroblastomas. Wild-type (WT) p53 is required for the activation of p53 signaling by Mdm2 inhibitors. This suggests that neuroblastoma could be amenable to intervention with Mdm2 inhibitors. In this study, we demonstrated that the ALK inhibitor, NVP-LDK378, in combination with a novel Mdm2 inhibitor, NVP-CGM097, promoted apoptosis in ALK mutant and p53 WT neuroblastoma cell lines. NVP-LDK378 inhibited ALK phosphorylation and NVP-CGM097 caused induction of p53 and its downstream target genes in these cell lines. Meanwhile, Mdm2 inhibition in MYCN-amplified neuroblastoma cell lines significantly decreased the levels of Mycn protein. In addition, NVP-LDK378 and NVP-CGM097 combination resulted in complete tumor regression and markedly prolonged survival in neuroblastoma xenograft models. Overall, NVP-LDK378 and NVP-CGM097 combination may provide an effective treatment for ALK mutant and p53 WT neuroblastoma patients.

Citation Format: Hui Qin Wang, Linda Battalagine, Jinsheng Liang, Ensar Halilovic, Robert Schlegel, Alan Huang, Z. Alexander Cao, John Monahan, Fang Li. The Mdm2 inhibitor NVP-CGM097 enhances the anti-tumor activity of NVP-LDK378 in ALK mutant neuroblastoma 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 2929. doi:10.1158/1538-7445.AM2014-2929
Abstract 2973: Mouse models of lung cancer mediated by lentiviral gene delivery
Authors: Xia Yifeng , Yeddula Narayana , Ke Eugene , Leblanc Mathias , Verma Inder
Published: May 2015

Lung cancer, the leading cause of cancer deaths, is responsible for 1.4 million lives lost worldwide every year. Good animal models that can faithfully recapitulate the human disease are in great need for pre-clinical studies. Based on the different responses to treatment, lung cancer is divided into two major classes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Adenocarcinoma (AdC) and squamous cell lung cancer (SqCC) are two major sub-types of NSCLC. We have successfully developed a novel lentiviral gene delivery system to study the initiation and development of all these lung cancers. We used CA2Cre-shp53 lentiviral vector to initiate lung adenocarcinoma in LSL-KrasG12D mice and proved the importance of NF-κB/IKK2 pathway in tumor cell proliferation. To search for novel tumor suppressors in lung cancer, we incorporated an shRNA library targeting ∼5000 cell signal genes into our CA2Cre lentivector and did the screening in the mice. We have identified several dozens of candidates and are validating these genes back in animals. SCLC is the most malignant form of lung cancer with a five-year survival less than 6%. A mouse model with p53/Rb deletions activated by Adeno-Cre has been established in Berns lab. To improve the model and study other genes that are frequently mutated in human SCLC, we designed a single lentiviral vector to deliver oncogenes (L-myc etc.) and shRNAs against tumor suppressors (p53, Rb etc) and successfully initiated SCLC in wild-type mice. Furthermore, we included luciferase or fluorescence protein genes in the same vector to follow tumor growth and metastasis in the live animal. Using this new model, we are now studying the crosstalk of multiple mutations in the SCLC development. SqCC has not been well modeled by genetic approaches. Interestingly, when we gave KrasG12D-shp53 lentiviral vector after lung tissue damage induced by naphthalene, the mice quickly developed squamous tumors in the big bronchi other than adenocarcinomas in the peripheral lung. Kras mutations are actually not common in human SqCC patients, so we are now optimizing the model and see if more relevant genetic changes such FGFR1 amplification can also induce this type of lung cancer in mice. We hope that our unique lentiviral tool can simplify and accelerate the lung cancer study to understand the big signal pathway complex leading to tumorigenesis and metastasis in the lung and search for new therapies for this deadly human disease.

Citation Format: Yifeng Xia, Narayana Yeddula, Eugene Ke, Mathias Leblanc, Inder Verma. Mouse models of lung cancer mediated by lentiviral gene delivery. [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 2973. doi:10.1158/1538-7445.AM2014-2973
Abstract 303: Cell Slider: Using crowd sourcing for the scoring of molecular pathology
Authors: Pharoah Paul D.
Published: May 2015

The advent of tumour microarrays has increased the throughput of pathology sample preparation but has led to a new bottleneck at the analysis stage as many thousands of samples need to be scored by an expert pathologist. We launched Cell Slider (www.clicktocure.net), the world's first Citizen Science project in cancer research, in Autumn 2012. Cell SliderTM aims to speed up the analysis of tumour microarray images through crowd sourcing. Citizen Scientists have now generated over 1.7 million scores of tumour images from the Breast Cancer Association Consortium image database.

In order to evaluate the validity of Citizen Scientist scoring we compared the scores of Citizen Scientists with those of an experienced pathologist for 2,575 tissue cores from the SEARCH study that have been stained for expression of the estrogen receptor. Each image of a 0.6mm tissue core stained for ER was divided into 12 sub-images, each of which was scored by five citizen scientists. A weighted average of the 60 (12x5) scores was used to generate a single citizen scientist score for each tissue core.

The average number of cancer cells observed by Citizen Scientists was used to indicate whether or not the core contained cancer. The sensitivity and specificity then depends on the threshold used for positivity. Of 2,575 cores on the TMA the pathologist identified 1,792 as having cancer tissue. The kappa statistic for agreement between pathologist and Citizen Scientist was 0.72. Using a threshold to give a sensitivity of 95 per cent, the specificity was 72 per cent.

We estimated the accuracy of assignment of tumour ER status using the 1,703 cores where both pathologist and Citizen Scientists identified cancer in the TMA core. A pseudo-Allred score on a continuous scale was calculated from the individual Citizen Scientist scores for the proportion of positively stained cells and the intensity of positively stained cells. The sensitivity and specificity of ER status depends on the threshold pseudo-Allred score used to determine ER positivity. The kappa statistic for agreement between pathologist and Citizen Scientist was 0.84. Using a threshold to give a sensitivity of 95 per cent, the specificity was 90 per cent.

We have shown that crowd sourcing can be used to score breast cancer TMAs stained by immunohistochemistry with a reasonable degree of accuracy. Further analysis are underway to determine whether accuracy can be improved using difference weighting schemes and to evaluate the accuracy of scoring of typical cytoplasmic and membrane molecular markers.

Citation Format: Paul D. Pharoah. Cell Slider: Using crowd sourcing for the scoring of molecular pathology. [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 303. doi:10.1158/1538-7445.AM2014-303
Abstract 4204: Isatin analog as bruton tyrosine kinase inhibitor: A promising novel agent for multiple myeloma treatment
Authors: Gowda Krishne , Pandey Manoj K. , Sharma Arun K. , Amin Shantu
Published: May 2015

Multiple myeloma (MM) is a clonal malignancy of plasma cells accumulating in the bone marrow. MM cells are highly dependent on the bone marrow (BM) microenvironment for growth and survival through interactions particularly with BM stromal cells (BMSCs), which secrete important MM growth factors and cytokines. Understanding and defining these BM factors are critical to provide the rationale to functionally target these factors and/or kinases as novel biologically based therapeutics for MM. Bruton tyrosine kinase (Btk), a non-receptor tyrosine kinase resembling the Src family, plays a key role in the development and function of normal B cells through activation of the B-cell antigen receptor signaling pathway on binding to antigens. Btk further phosphorylates PLC-γ, leading to activation of MAPK, NF-κB, and AKT signaling pathways. Earlier our group synthesized a novel series of 5,7-dibromoisatin analogs. Analogs 6, 11 and 13 were potential anti-cancer agents. In the present study, through docking studies we predicted that bromo isatin analog-11 directly interacts with Btk. We found that Isatin-11 inhibits Btk activity in a dose- and time-dependent manner. Isatin-11 down-regulates the expression of Bcl-2, Bcl-xL on MM cells. Isatin-11 also activates caspase-3 and PARP-cleavage in MM cells. Taken together, these results suggest that targeting of Btk by Isatin-11 may represent a new therapeutic strategy to treat MM.

Citation Format: Krishne Gowda, Manoj K. Pandey, Arun K. Sharma, Shantu Amin. Isatin analog as bruton tyrosine kinase inhibitor: A promising novel agent for multiple myeloma treatment. [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 4204. doi:10.1158/1538-7445.AM2014-4204
Abstract 4229: The anti-estrogen effect of metformin in ERα+ breast cancer and tamoxifen resistant cell lines
Authors: Kim Jinkyoung , Lee Jiyun , Jang Soonyoung , Kim Chungyeul , Kim Aeree
Published: May 2015

Anti-estrogen therapy is still the most important systemic treatment to manage estrogen responsive breast cancer. Tamoxifen resistance is a major obstacle in treatment of ER-positive breast cancer. Recently, metformin, which is a biguanide derivative which is commonly used in the treatment of type II diabetes, has been emerged as a potential anti-cancer agent. We investigated if metformin had anti-estrogen effect in ER-positive breast cancer cell lines which are MCF-7, MDA-MB-361 as well as tamoxifen resistant cells (TR-MCF-7). Metformin inhibited the cell proliferation and the expression of ERα in dose-dependent manner in MCF-7 and TR-MCF-7 cells. Under stimulation of 17β-estradiol (E2), metformin inhibited the cell proliferation and expression of mRNA and protein of ERα in MCF-7 and TR-MCF-7 cells, while tamoxifen didn't affect E2 induced cell proliferation and ERα expression. This result indicates that metformin, as anti-estrogen agent, is superior to tamoxifen for targeting ER in breast cancer. We also identified that metformin inhibited E2 induced cell proliferation and expression of mRNA and protein of ERα in MDA-MB 361 cell line (ER+/Her-2+). We also analyzed estrogen responsive element (ERE) activity using by luciferase assay and identified metformin suppressed E2-inducible luciferase activity and expression of direct ER-regulated genes, such as c-myc, Cyclin D1 and pS2 and FOXA1, which were not inhibited by tamoxifen. In conclusion, metformin has a direct anti proliferative effect and anti-estrogen effect in MCF-7, TR-MCF-7 as well as MDA-MB-361 cell lines. These results suggest that metformin can be used as anti- estrogen in ER positive breast cancer (luminal A & B) and tamoxifen resistant breast cancer.

Citation Format: Jinkyoung Kim, Jiyun Lee, Soonyoung Jang, Chungyeul Kim, Aeree Kim. The anti-estrogen effect of metformin in ERα+ breast cancer and tamoxifen resistant cell lines. [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 4229. doi:10.1158/1538-7445.AM2014-4229
Abstract 681: Heat shock-mediated suppression of ODC protein expression in colorectal cancer cells
Authors: Wales Christina T.K. , Jacobs Aaron T.
Published: May 2015

Polyamines are small, positively charged metabolites that have important roles in several cancer hallmarks, including cell proliferation, invasion, and angiogenesis. Difluoromethylornithine (DFMO) is an FDA-approved drug that is in clinical trials for treatment of colorectal cancer. DFMO functions by inhibiting ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine biosynthesis. By reducing polyamine levels, DFMO has shown much promise as an anti-cancer chemotherapeutic. One potential limitation to DFMO is that it causes a profound increase in ODC expression, which we suspect may counteract the intended use of the drug. However, strategies to suppress ODC expression would theoretically increase DFMO efficacy.

We have discovered that transient hyperthermia (heat shock, 42°C) causes a profound and rapid suppression in ODC protein levels in RKO colorectal cancer cells. To investigate the responsible mechanism, we began by examining ODC mRNA expression and stability. Hyperthermia caused only a moderate reduction in ODC transcript levels. Also, mRNA stability, which was assessed using actinomycin D, was not reduced by heat shock. We next examined the effect of hyperthermia on the control of ODC protein turnover, which is known to be mediated by ODC antizyme (OAZ1). We performed real-time PCR to assess the effect of heat shock on OAZ1 expression. Data reveal a significant increase in the level of OAZ1 transcript. Since heat shock is known to activate the transcription factor HSF1, we next investigated the role of HSF1 in OAZ1 induction. Using siRNA to silence HSF1, we found that the induction of OAZ1 by heat shock is fully dependent on HSF1 expression. In addition to heat shock, HSF1 is also activated by various chemotherapeutics, but most notably by Hsp90 inhibitors. We therefore tested if Hsp90 inhibitors, geldanamycin and 17-AAG could be used to induce OAZ1 and promote ODC turnover. These results show that both Hsp90 inhibitors caused a significant activation of HSF1, an induction in OAZ1 expression, and decrease in ODC protein levels. We therefore propose that Hsp90 inhibitors may be effective in attenuating ODC expression and polyamine metabolism. We suggest that they may be used in combination with DFMO to prevent the compensatory increase in ODC levels and increase anti-cancer effects.

Citation Format: Christina T.K. Wales, Aaron T. Jacobs. Heat shock-mediated suppression of ODC protein expression 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 681. doi:10.1158/1538-7445.AM2014-681
Abstract 832: Pre-clinical development of EC1456: A potent Folate targeted Tubulysin SMDC
Authors: Reddy Joseph A. , Bloomfield Alicia , Nelson Melissa , Dorton Ryan , Vetzel Marilynn , Leamon Christopher P.
Published: May 2015

Folate targeted small molecule drug conjugates (SMDC) have shown promising results in early stage clinical trials with Vintafolide now being evaluated in a phase 3 trial in FR-positive ovarian cancer patients. We have now developed a new group of folate conjugated Tubulysins, of which EC1456 has emerged as a lead candidate for clinical development. Treatment of nude mice bearing folate receptor (FR) positive human xenografts with EC1456 led to complete remissions (CR's) or cures in 100% of the mice at various doses and schedules. The observed activity was not accompanied by any noticeable weight loss or major organ tissue degeneration. In contrast, no significant anti-tumor activity (0 % CR's) was observed in EC1456-treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating EC1456's target-specific activity. The enhanced therapeutic index due to folate conjugation was also evidenced by the fact that the un-targeted free drug (tubulysin B or its hydrazide form) was found to be completely inactive even when administered at highly toxic dose levels. Furthermore, when challenged with larger tumors, EC1456 again displayed remarkable anti-tumor activity with 100% cures in tumors up to 750 mm3. Complete cures were also observed in other FR positive models such as MDA-MB-231 TNBC and M109 lung tumor models. In support of our clinical strategy to treat drug resistant FR-positive cancer patients we have also shown that EC1456 was highly active against FR expressing paclitaxel and cisplatin-resistant cell lines. Taken together, these studies demonstrated that EC1456 has significant anti-tumor growth activity and tolerability, thus lending support to the ongoing Phase 1 clinical evaluation of EC1456 for advanced malignancies.

Citation Format: Joseph A. Reddy, Alicia Bloomfield, Melissa Nelson, Ryan Dorton, Marilynn Vetzel, Christopher P. Leamon. Pre-clinical development of EC1456: A potent Folate targeted Tubulysin SMDC. [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 832. doi:10.1158/1538-7445.AM2014-832
Abstract P6-05-01: Endoplasmic reticulum resident protein transmembrane protein 33 (TMEM33) induces apoptosis via UPR signaling and autophagy in breast cancer cells
Authors: Hu Rong , Zhang Xiyuan , Hilakivi-Clarke Leena , Kasid Usha , Clarke Robert
Published: May 2015

Breast cancer is the most common cancer type in women, with expected incidence of around 232,670 new cases in the US alone each year. Endoplasmic reticulum stress (EnR stress) and the related unfolded protein response (UPR) are activated in breast cancer cells to promote cell survival and endocrine resistance. TMEM33 is a novel transmembrane protein that resides in the endoplasmic reticulum (EnR) and has been shown to activate two of the UPR branches (PERK; IRE1α). However, the cellular functions and underlying mechanism of this EnR resident protein remains largely unknown. In this study, we show that overexpression of TMEM33 induces robust cell death in breast cancer cells. TMEM33 overexpression strongly activates the pro-death JNK-p53 pathway, possibly through the activation of IRE1α. We also observe a significant inhibition of the downstream survivin (also called BIRC5), which is known to inhibit cell death by binding to caspases and blocking their activation. We further show that either the blockage of JNK activation with a small molecule inhibitor, or the overexpression of survivin, protects cells against TMEM33-induced apoptosis. In addition, we demonstrate that TMEM33 overexpression induces autophagy in breast cancer cells (changes in LC3 and p62 expression). Inhibition of autophagy with either the autophagy inhibitor chloroquine, or by knockdown of the autophagy gene Atg5, further sensitizes breast cancer cells to TMEM33 overexpression. Conversely, cell death induced by TMEM33 is decreased by overexpression of the autophagy gene Beclin 1. The findings in this study demonstrate that the novel EnR resident protein TMEM33 induces cell death by activating the IRE1α-JNK-p53-survivin pathway in breast cancer cells. Paradoxically, autophagy is also activated by TMEM33 but the prosurvival action is generally not sufficient to block all of the cell death signaling.

Citation Format: Rong Hu, Xiyuan Zhang, Leena Hilakivi-Clarke, Usha Kasid, Robert Clarke. Endoplasmic reticulum resident protein transmembrane protein 33 (TMEM33) induces apoptosis via UPR signaling and autophagy in breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-05-01.
Sprouty2 Drives Drug Resistance and Proliferation in Glioblastoma
Authors: Walsh Alice M. , Kapoor Gurpreet S. , Buonato Janine M. , Mathew Lijoy K. , Bi Yingtao , Davuluri Ramana V. , Martinez-Lage Maria , Simon M. Celeste , O'Rourke Donald M. , Lazzara Matthew J.
Published: May 2015

Glioblastoma multiforme (GBM) is notoriously resistant to therapy, and the development of a durable cure will require the identification of broadly relevant regulators of GBM cell tumorigenicity and survival. Here, we identify Sprouty2 (SPRY2), a known regulator of receptor tyrosine kinases (RTK), as one such regulator. SPRY2 knockdown reduced proliferation and anchorage-independent growth in GBM cells and slowed xenograft tumor growth in mice. SPRY2 knockdown also promoted cell death in response to coinhibition of the epidermal growth factor receptor (EGFR) and the c-MET receptor in GBM cells, an effect that involved regulation of the ability of the p38 mitogen-activated protein kinase (MAPK) to drive cell death in response to inhibitors. Analysis of data from clinical tumor specimens further demonstrated that SPRY2 protein is definitively expressed in GBM tissue, that SPRY2 expression is elevated in GBM tumors expressing EGFR variant III (EGFRvIII), and that elevated SPRY2 mRNA expression portends reduced GBM patient survival. Overall, these results identify SPRY2 and the pathways it regulates as novel candidate biomarkers and therapeutic targets in GBM.

Implications: SPRY2, counter to its roles in other cancer settings, promotes glioma cell and tumor growth and cellular resistance to targeted inhibitors of oncogenic RTKs, thus making SPRY2 and the cell signaling processes it regulates potential novel therapeutic targets in glioma. Mol Cancer Res; 13(8); 1227–37. ©2015 AACR.
miR-19–Mediated Inhibition of Transglutaminase-2 Leads to Enhanced Invasion and Metastasis in Colorectal Cancer
Authors: Cellura D. , Pickard K. , Quaratino S. , Parker H. , Strefford J.C. , Thomas G.J. , Mitter R. , Mirnezami A.H. , Peake N.J.
Published: May 2015

Transglutaminase-2 (TG2) is a critical cross-linking enzyme in the extracellular matrix (ECM) and tumor microenvironment (TME). Although its expression has been linked to colorectal cancer, its functional role in the processes that drive disease appears to be context dependent. There is now considerable evidence of a role for microRNAs (miRNA) in the development and progression of cancer, including metastasis. A cell model of metastatic colon adenocarcinoma was used to investigate the contribution of miRNAs to the differential expression of TG2, and functional effects on inflammatory and invasive behavior. The impact of TG2 in colorectal cancer was analyzed in human colorectal tumor specimens and by manipulations in SW480 and SW620 cells. Effects on invasive behavior were measured using Transwell invasion assays, and cytokine production was assessed by ELISA. TG2 was identified as a target for miR-19 by in silico analysis, which was confirmed experimentally. Functional effects were evaluated by overexpression of pre-miR-19a in SW480 cells. Expression of TG2 correlated inversely with invasive behavior, with knockdown in SW480 cells leading to enhanced invasion, and overexpression in SW620 cells the opposite. TG2 expression was observed in colorectal cancer primary tumors but lost in liver metastases. Finally, miR-19 overexpression and subsequent decreased TG2 expression was linked to chromosome-13 amplification events, leading to altered invasive behavior in colorectal cancer cells.

Implications: Chromosome-13 amplification in advanced colorectal cancer contributes to invasion and metastasis by upregulating miR-19, which targets TG2. Mol Cancer Res; 13(7); 1095–105. ©2015 AACR.
Colorectal Carcinogenesis: Connecting K-RAS–Induced Transformation and CREB Activity In Vitro and In Vivo
Authors: Steven André , Heiduk Max , Recktenwald Christian V. , Hiebl Bernhard , Wickenhauser Claudia , Massa Chiara , Seliger Barbara
Published: May 2015

Oncogenic transformation is often associated with an increased expression of the cAMP response element binding (CREB) transcription factor controlling the expression of genes involved in cell proliferation, cell cycle, apoptosis, and tumor development, but a link between K-RASV12-induced transformation and CREB has not yet been determined. Therefore, the constitutive and/or inhibitor-regulated mRNA and protein expression of CREB and signal transduction components and growth properties of parental fibroblasts, K-RASV12-transformed counterparts, shCREB K-RASV12 transfectants and human colon carcinoma cells were determined. Increased CREB transcript and protein levels accompanied by an enhanced CREB activity was detected in K-RASV12-transformed murine fibroblasts and K-RASV12-mutated human tumor cells, which is dependent on the MAPK/MEK, PI3K, and/or PKC signal transduction. Immunohistochemical (IHC) staining of colorectal carcinoma lesions and murine tumors, with known KRAS gene mutation status, using antibodies specific for CREB and phospho-CREB, revealed a mechanistic link between CREB expression and K-RASV12-mutated colorectal carcinoma lesions when compared with control tissues. Silencing of CREB by shRNA and/or treatment with a CREB inhibitor (KG-501) reverted the neoplastic phenotype of K-RASV12 transformants as demonstrated by a more fibroblast-like morphology, enhanced apoptosis sensitivity, increased doubling time, decreased migration, invasion and anchorage-independent growth, reduced tumorigenesis, and enhanced immunogenicity in vivo. The impaired shCREB-mediated invasion of K-RASV12 transformants was accompanied by a transcriptional downregulation of different matrix metalloproteinases (MMP) coupled with their reduced enzymatic activity.

Implications: CREB plays a key role in the K-RASV12-mediated neoplastic phenotype and represents a suitable therapeutic target for murine and human K-RASV12-induced tumors. Mol Cancer Res; 13(8); 1248–62. ©2015 AACR.
Transglutaminase Is Required for Epidermal Squamous Cell Carcinoma Stem Cell Survival
Authors: Fisher Matthew L. , Keillor Jeffrey W. , Xu Wen , Eckert Richard L. , Kerr Candace
Published: May 2015

Cancer stem cells are thought to be responsible for rapid tumor growth, metastasis, and enhanced tumor survival following drug treatment. For this reason, there is a major emphasis on identifying proteins that can be targeted to kill cancer stem cells or control their growth, and transglutaminase type II (TGM2/TG2) is such a target in epidermal squamous cell carcinoma. TG2 was originally described as a transamidase in the extracellular matrix that crosslinks proteins by catalyzing ϵ-(γ-glutamyl)lysine bonds. However, subsequent studies have shown that TG2 is a GTP-binding protein that plays an important role in cell signaling and survival. In the present study, TG2 shows promise as a target for anticancer stem cell therapy in human squamous cell carcinoma. TG2 was determined to be highly elevated in epidermal cancer stem cells (ECS cells), and TG2 knockdown or suppression of TG2 function with inhibitors reduced ECS cell survival, spheroid formation, Matrigel invasion, and migration. The reduction in survival is associated with activation of apoptosis. Mechanistic studies, using TG2 mutants, revealed that the GTP-binding activity is required for maintenance of ECS cell growth and survival, and that the action of TG2 in ECS cells is not mediated by NF-κB signaling.

Implications: This study suggests that TG2 has an important role in maintaining cancer stem cell survival, invasive, and metastatic behavior and is an important therapeutic target to reduce survival of cancer stem cells in epidermal squamous cell carcinoma. Mol Cancer Res; 13(7); 1083–94. ©2015 AACR.
Defective Myb Function Ablates Cyclin E1 Expression and Perturbs Intestinal Carcinogenesis
Authors: Cheasley Dane , Pereira Lloyd , Sampurno Shienny , Sieber Oliver , Jorissen Robert , Xu Huiling , Germann Markus , Yuqian Yan , Ramsay Robert G. , Malaterre Jordane
Published: May 2015

Cyclin E1 is essential for the reentry of quiescent cells into the cell cycle. When hypomorphic mutant Myb mice (MybPlt4) were examined, it was noted that Cyclin E1 (Ccne1) expression was reduced. Furthermore, the induction of Ccne1 in recovering intestinal epithelia following radiation-induced damage was ablated in Myb-mutant mice. These data prompted us to investigate whether Myb directly regulated Ccne1 and to examine whether elevated Myb in colorectal cancer is responsible for Cyclin E1–driven tumor growth. Here, it was found that Myb/MYB and Ccne1/CCNE1 expressions were coupled in both mouse and human adenomas. In addition, the low molecular weight Cyclin E1 was the predominant form in intestinal crypts and adenomatous polyposis coli (Apc)–mutant adenomas. Chromatin immunoprecipitation (ChIP) analysis confirmed that Myb bound directly to the Ccne1 promoter and regulated its endogenous expression. In contrast, MybPlt4 served as a dominant-negative factor that inhibited wild-type Myb and this was not apparently compensated for by the transcription factor E2F1 in intestinal epithelial cells. MybPlt4/Plt4 mice died prematurely on an ApcMin/+ background associated with hematopoietic defects, including a myelodysplasia; nevertheless, ApcMin/+ mice were protected from intestinal tumorigenesis when crossed to MybPlt4/+ mice. Knockdown of CCNE1 transcript in murine colorectal cancer cells stabilized chromosome ploidy and decreased tumor formation. These data suggest that Cyclin E1 expression is Myb dependent in normal and transformed intestinal epithelial cells, consistent with a cell-cycle progression and chromosome instability role in cancer.

Implications: This study demonstrates that Myb regulates Cyclin E1 expression in normal gastrointestinal tract epithelial cells and is required during intestinal tumorigenesis. Mol Cancer Res; 13(8); 1185–96. ©2015 AACR.
HIC1 Tumor Suppressor Loss Potentiates TLR2/NF-κB Signaling and Promotes Tissue Damage–Associated Tumorigenesis
Authors: Janeckova Lucie , Pospichalova Vendula , Fafilek Bohumil , Vojtechova Martina , Tureckova Jolana , Dobes Jan , Dubuissez Marion , Leprince Dominique , Baloghova Nikol , Horazna Monika , Hlavata Adela , Stancikova Jitka , Sloncova Eva , Galuskova Katerina , Strnad Hynek , Korinek Vladimir
Published: May 2015

Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses “danger” signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells.

Implications: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells. Mol Cancer Res; 13(7); 1139–48. ©2015 AACR.
Abstract A003: Darpp-32 and t-Darpp are differentially expressed in normal and malignant mouse mammary tissue
Authors: Christenson Jessica L. , Susan Kane E.
Published: May 2015

Darpp-32 (dopamine and cAMP regulated phosphoprotein of 32 kD) and t-Darpp are expressed in various types of cancer, including several forms of breast cancer. Both are transcribed from the gene PPP1R1B (Protein phosphatase 1, regulatory (inhibitor) subunit 1B) via alternative promoters, t-Darpp being an N-terminally truncated version of the full length protein, Darpp-32. Although Darpp-32 is a well-known regulator of neurotransmission, its role in cancer is less well understood. t-Darpp is believed to be a putative oncogene: increasing cellular growth, inhibiting apoptosis and contributing to acquired drug resistance. In humans, Darpp-32 is found in both normal and malignant breast tissue, whereas t-Darpp has only been found in malignant breast tissue. Overall, however, the exact biological functions of these proteins in normal and malignant breast tissues are not well understood, and to date they have only been examined in patient samples, cell lines and xenograft mouse models. The use of transgenic mouse mammary tumor models, such as MMTV-PyVT and MMTV-Neu, to study the roles of Darpp-32 and t-Darpp in breast cancer in vivo have been limited by the lack of knowledge regarding t-Darpp expression in mice. In this study, we investigated Darpp-32 and t-Darpp mRNA and protein expression in normal and malignant mouse mammary tissue. We found that Darpp-32 was expressed in normal mouse mammary tissue and in some breast tumors, whereas t-Darpp was found only in the malignant mammary tissue. t-Darpp was expressed in the majority of breast tumors often at equal or higher levels than Darpp-32. t-Darpp expression did not seem to be correlated with either tumor location or tumor size. This shift in expression from Darpp-32 to t-Darpp during mouse mammary malignant transformation is reminiscent of the expression patterns observed in human breast tissue, suggesting that transgenic mouse mammary tumor models might be an ideal tool for future investigation of Darpp-32 and t-Darpp in breast cancer.

Citation Format: Jessica L. Christenson, Kane E. Susan. Darpp-32 and t-Darpp are differentially expressed in normal and malignant mouse mammary tissue. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A003.