bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4355: Inhibition of RNA Polymerase I as a strategy to treat cancer.
Authors: Bywater Megan J. , Hannan Katherine M. , Poortinga Gretchen , Devlin Jennifer R. , Cullinane Carleen , Drygin Denis , Rice William G. , Von Hoff Daniel , Johnstone Ricky W. , McArthur Grant A. , Hannan Ross D. , Pearson Richard B.
Published: August 2014

Abnormalities of the nucleolus, the site of transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I), have been recognized as diagnostic for cancer for more then a century. However, a critical, unresolved question has been whether the accelerated ribosome biogenesis responsible for the nucleolar changes is required for malignancy.

Here we show that the PI3K/AKT pathway, deregulated in a high proportion of human tumours, is a critical regulator of ribosome biogenesis. Active AKT is sufficient to drive rRNA synthesis and ribosome biogenesis. Furthermore, AKT cooperates with c-MYC to drive these processes identifying the AKT//MYC network as a master controller of cell growth. Consistent with this concept, AKT activity is required for maximal activation of rRNA synthesis and tumour formation in the Eμ-Myc mouse model of Burkitt's lymphoma (1). Our findings raise the possibility that cancers characterized by unrestrained cellular growth may be vulnerable to therapeutic strategies that target ribosome biogenesis.

To directly test this hypothesis, we used a novel selective small molecule inhibitor of Pol I transcription (CX-5461) (2), to show that Pol I can be targeted in vivo to treat tumors in mouse models of lymphoma and leukemia through the activation of p53-dependent apoptosis, while sparing normal hematologic cells. Thus, selective inhibition of Pol I transcription can serve as a novel therapeutic strategy for the treatment of cancer (3). A Phase 1 trial of this first-in-class molecule begins in 2013 at the Peter MacCallum Cancer Centre for patients with haematologic malignancies. Strikingly, allosteric inhibitors of AKT suppress rRNA synthesis independent of p53 and cooperate with CX-5461 in killing Eμ-Myc lymphomas providing a clear rationale for combining these agents in future trials.

Citation Format: Megan J. Bywater, Katherine M. Hannan, Gretchen Poortinga, Jennifer R. Devlin, Carleen Cullinane, Denis Drygin, William G. Rice, Daniel Von Hoff, Ricky W. Johnstone, Grant A. McArthur, Ross D. Hannan, Richard B. Pearson. Inhibition of RNA Polymerase I as a strategy to treat cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4355. doi:10.1158/1538-7445.AM2013-4355
Abstract 4360: Involvement of intermediate filament nestin in cell growth of small-cell lung cancer.
Authors: Kunii Eiji , Takakuwa Osamu , Maeno Ken , Oguri Tetsuya , Yokoyama Midori , Hijikata Hisatoshi , Uemura Takehiro , Kasai Daishi , Ohkubo Hirotsugu , Miyazaki Mikinori , Niimi Akio
Published: August 2014

Small-cell lung cancer (SCLC) represents 13% of all newly diagnosed cases of lung cancer. Clinical progression of SCLC is rapid and aggressive. Unfortunately, 70% of SCLC cases have already metastasized at the time of diagnosis, and the recent median survival time and two-year survival rate in SCLC patients with extensive-stage disease is only 8-13 months and 5%, respectively. Clinical trials of SCLC treatments have been conducted since the mid-1980s, but have not achieved prolonged survival; this lack of success is likely due to a lack of appropriate target molecules. To improve outcomes for SCLC patients, new therapeutic strategies including novel molecular targets for SCLC are desired. Nestin is a class VI intermediate filament protein expressed in stem/progenitor cells during the development of the central nervous system. Nestin is detected in various types of tumors and reported to be involved in malignant phenotypes. Here, we investigated the expression and function of nestin in SCLC. At the beginning, protein expression of nestin and achaete-scute homolog 1 (ASH1) was studied in 21 lung cancer cell lines. Nestin was expressed in 9 of 10 SCLC cell lines. The nestin expression level in SCLC cell lines was significantly higher than that in non-small-cell lung cancer (NSCLC) cell lines (P<0.01). There was a statistically significant positive correlation between the expression levels of nestin and ASH1 in SCLC cell lines, but not in NSCLC cell lines. To assess the function of nestin, we transfected a short hairpin RNA (shRNA) targeting nestin into two SCLC cell lines, DMS53 and SBC3, and then obtained cloned cells that showed apparent down-regulation of nestin. Nestin knock-down cells created by transfection with shRNA exhibited decreased invasion and cell proliferation capabilities. In conclusion, Nestin is expressed in SCLC in association with neuro-endocrine feature and participates in malignant phenotypes including cell growth. Therefore, nestin may be a novel therapeutic target of SCLC. Additional in vivo study will be needed.

Citation Format: Eiji Kunii, Osamu Takakuwa, Ken Maeno, Tetsuya Oguri, Midori Yokoyama, Hisatoshi Hijikata, Takehiro Uemura, Daishi Kasai, Hirotsugu Ohkubo, Mikinori Miyazaki, Akio Niimi. Involvement of intermediate filament nestin in cell growth of small-cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4360. doi:10.1158/1538-7445.AM2013-4360
Abstract 4364: Copy number gain of PTK2 and PIK3CA mutations are common genetic alterations in ovarian clear cell carcinoma.
Authors: Yoon Heejei , Choi Yoon-La , Song Ji-Young , Do Ingu , Kang So Young , Ko Young-Hyeh , Song Sangyong , Kim Byong-Gie
Published: August 2014

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer and accounts for 5-25% of ovarian cancers. OCCC has shown higher resistance to first-line platinum and taxane-based chemotherapy, resulting in worse prognosis. Most common mutations occur in the genes ARID1A (46%) and PIK3CA (33%). Using array comparative genomic hybridization (aCGH), we previously identified frequent chromosomal gain at 8q24, which includes the encoding sequence for focal-adhesion kinase (FAK)/Protein Tyrosine Kinase 2 (PTK2), a protein that provides signaling and scaffolding functions at sites of integrin adhesion. To validate the copy number gain of PTK2 and correlate the copy number variations with PIK3CA mutations, we analyzed the copy number gain and protein expression levels of PTK2, and examined the PIK3CA mutations in 67 OCCC samples. We found that PTK2 copy number gain and loss were at a frequency of 23% and 25%, respectively. PTK2 gain significantly correlated with the strong expression of PTK2. PIK3CA mutations were found in 20% of the samples. About 40% of the tested samples showed PTK2 gain or PIK3CA mutation. PIK3CA mutations are not strictly mutually exclusive to PTK2 gain, but are more likely to occur in samples with PTK2 copy number normal or loss. Genetic alterations in OCCC mainly activate the PTK2/PI3K/AKT signaling pathway, which makes the pathway an attractive candidate for targeted therapeutics. We, therefore, treated OVMANA, OVISE and OVTOKO OCCC cell lines with PTK2 inhibitors (PF-562271 or PF-573228), or a combination of PF-562271 with a PI3K/mTOR dual inhibitor (BEZ235). Copy number variations of PTK2 were not related to the drug sensitivity of the OCCC cell lines. Drug sensitivity of most of the cells was enhanced toward PF-562271 than PF-573228. OVMANA cells with PIK3CA mutations were more sensitive to a combination of PI3K/mTOR and PTK2 inhibitor than the cells without PIK3CA mutations. Our findings thus suggest that the PTK2/PI3K/AKT signaling pathway is frequently activated in OCCC, and a combination of inhibitor drugs would prove to be necessary to enhance the overall drug efficacy in treating OCCC.

Citation Format: Heejei Yoon, Yoon-La Choi, Ji-Young Song, Ingu Do, So Young Kang, Young-Hyeh Ko, Sangyong Song, Byong-Gie Kim. Copy number gain of PTK2 and PIK3CA mutations are common genetic alterations in ovarian clear cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4364. doi:10.1158/1538-7445.AM2013-4364
Abstract 4366: ShRNA-based cellular proliferation signaling analysis revealed DRD2 as a novel therapeutic target for glioblastoma.
Authors: Li Jie , Zhu Shan , Kozono David , Futulan Diahnn , Gonda David , Kushwaha Deepa , Kahle Kristopher , Elledge Stephen , Chen Clark C.
Published: August 2014

Glioblastoma remains one of the deadliest of human cancers, with most patients succumbing to the disease within one year of diagnosis. Our best current understanding of its etiology is that glioblastoma involves genetic and epigenetic changes that subvert the inherent molecular circuitry of glial cells to drive uncontrolled cellular proliferation. Unfortunately, this molecular circuitry in glioblastoma appears sufficiently complex and redundant that disruption at any particular point tends to redirects signaling through the remaining circuit. In this context, meaningful therapeutic gains will require the identification of therapeutic agents that target critical nodes within this circuit. To identify novel therapeutic agents that target critical nodes which regulate cellular proliferation, a genome-wide retroviral shRNA library screen was conducted in two glioblastoma cell lines (U87MG and A172) and two lung carcinoma lines (A549 and NCI-H460). Pathway analysis of the pro-proliferative genes using both PANTHER and Ingenuity software revealed an over-representation of G-protein coupled neurotransmitter receptors (GPCR), including dopamine receptor subtype 2 (DRD2), which was also highly expressed in glioblastoma specimens relative to the matched normal parentchymal. Further investigation demonstrated that DRD2 signal regulates cell growth by GNAI2-induced ERK activation. Moreover, Haloperidol, one of DRD2 antagonists, enhances significantly anti-tumor efficacy of EGFR inhibitor AG1478 using U87 and mesenchymal neuroshpere xenografy mouse models. Given that both AG1478 and DRD2 antagonists have been approved for the treatment of glioblastoma and psychotics, separtately, Haloperidol combined with chemotherapy represents a novel and likely conducted strategy for the treatment of glioblastoma.

Citation Format: Jie Li, Shan Zhu, David Kozono, Diahnn Futulan, David Gonda, Deepa Kushwaha, Kristopher Kahle, Stephen Elledge, Clark C. Chen. ShRNA-based cellular proliferation signaling analysis revealed DRD2 as a novel therapeutic target for glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4366. doi:10.1158/1538-7445.AM2013-4366
Abstract 4367: Differential contribution of chromatin remodeling complex components to the protection from cytotoxic stress.
Authors: Freeman Miles D. , Mazu Tryphon , Flores-Rozas Hernan
Published: August 2014

Anthracyclines are among the most common chemotherapeutic agents used to-date. Although effective, resistance to this agent usually develops in the course of treatment and serious dose-related toxicity, namely chronic cardio-toxicity, limits its use. In patients suffering from triple negative breast cancer (TNBC) cytotoxic agents such as anthracyclines are some of the only therapeutic alternatives. 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 anthracylcines. Of these, 11 genes are involved in chromatin remodeling. The most sensitive mutations corresponded to swi3, snf2 and arp8, which are components of the SWI/SNF complex. Deletion of SNF2 resulted in sensitivity to doxorubicin of 100,000-fold higher than wild type cells. Further analysis of the SNF2 defective strain revealed that it is also sensitive to daunorubicin and cisplatin, as well as to other DNA damaging agents such as alkylating agents, the topoI inhibitor campthotecin or replication blocks from hydroxyurea. Our results indicate that the SWI/SNF complex plays a critical role in protecting cells from exposure to anthracyclines and other DNA damaging agents. Targeting these genes may provide a useful alternative to hypersensitize cancer cells to chemotherapy and provide a therapeutic alternative to TNBC. Research supported by NIH/NCRR RCMI grant #RR03020 at Florida A&M University.

Citation Format: Miles D. Freeman, Tryphon Mazu, Hernan Flores-Rozas. Differential contribution of chromatin remodeling complex components to the protection from cytotoxic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4367. doi:10.1158/1538-7445.AM2013-4367
Abstract 4368: Cathepsin B is a potential therapeutic target in pancreatic and gastric cancer .
Authors: Wang Mei , Huang Chunlei , Dong Harvey , Qian Ziliang , Fan Shuqiong , Su Xinying , Zheng Li , Ji Qunsheng , Yu De-Hua
Published: August 2014

Proteases are critical in tumorigenesis by facilitating rapid cell cycling, mediating local invasion, fueling angiogenesis, and promoting metastasis. Cathepsin B (CTSB), a member of papain subfamily of lysosomal cysteine proteases, is involved in tumor cell invasion, metastasis, and angiogenesis. Emerging evidence indicated that CTSB is up-regulated in many cancer nodes and metastatic lesions and functions to protect tumor cells from apoptosis. Hererin, we have performed microarray-based comparative hybridization (aGCH) to screen gene copy number variations in primary tumor samples and found CTSB gene is amplified in 13% (14/107) of Chinese gastric cancer and 2% (1/54) of Chinese pancreatic samples. Theses amplifications were confirmed by fluorescence in situ hybridization (FISH) analysis. By using siRNA-mediated gene silencing, we demonstrated for the first time a role of CTSB in cancer cell proliferation. Down-regulation of CTSB by siRNA interference resulted in significantly reduced cell proliferation and increased cell death in HupT4, a CTSB amplified human pancreatic cancer cell line, but not in the non-amplified cell lines. Together, our results suggest that CTSB is a potential therapeutic target for pancreatic and gastric cancer with CTSB amplification.

Citation Format: Mei Wang, Chunlei Huang, Harvey Dong, Ziliang Qian, Shuqiong Fan, Xinying Su, Li Zheng, Qunsheng Ji, De-Hua Yu. Cathepsin B is a potential therapeutic target in pancreatic and gastric cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4368. doi:10.1158/1538-7445.AM2013-4368
Abstract 4371: Anti-leukemia effect of Casein Kinase II (CK2) inhibitors in vivo is mediated by the Ikaros tumor suppressor.
Authors: Dovat Sinisa , Song Chunhua , Payne Kimberly J.
Published: August 2014

An increase in the expression and activity of pro-oncogenic Casein Kinase II (CK2) is associated with the development of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) in humans. The mechanism by which CK2 promotes leukemogenesis is unknown. Previously, we demonstrated that CK2 kinase phosphorylates the Ikaros tumor suppressor in vivo and that phosphorylation of Ikaros at specific amino acids interferes with the normal function of the Ikaros protein. We hypothesized that CK2 exerts its pro-oncogenic effect by reducing the tumor suppressor activity of Ikaros and that inhibition of CK2 could enhance Ikaros tumor suppressor activity in leukemia. Here we show that Ikaros binds to the promoter regions and represses transcription of several genes that promote cell cycle progression in leukemia. Inhibition of CK2 results in: 1) enhanced Ikaros DNA-binding affinity to its target genes as indicated by quantitative chromatin immunoprecipitation (qChIP) assay); 2) increased Ikaros-mediated transcriptional repression of Ikaros target genes as demonstrated by luciferase reporter assay; 3) impaired proliferation of leukemia cells and 4) increased sensitivity of leukemia cells to Doxorubicin. The use of specific CK2 inhibitors in vivo in a human-mouse leukemia xenograft model produced a strong anti-leukemia effect. Functional analysis of leukemia cells from xenografts demonstrated that CK2 inhibition results in strong binding of Ikaros at the promoters of its target genes and reduced transcription of these genes as indicated by quantitative real-time PCR (qRT-PCR). Our results provide evidence that CK2 inhibitors have a potent anti-leukemia effect in vitro and in vivo, and suggest the mechanism through which CK2 inhibitors exert their therapeutic effect. The presented data demonstrate that one of the mechanisms of action for CK2 inhibitors involves increasing Ikaros activity as a transcriptional repressor in leukemia cells, resulting in enhanced tumor suppressor activity. These results provide a rationale for the use of CK2 inhibitors as a novel therapeutic modality for leukemia. Supported by National Institutes of Health R01 HL095120, a St. Baldrick's Foundation Career Development grant, and the Four Diamonds Fund Endowment (to S.D.).

Citation Format: Sinisa Dovat, Chunhua Song, Kimberly J. Payne. Anti-leukemia effect of Casein Kinase II (CK2) inhibitors in vivo is mediated by the Ikaros tumor suppressor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4371. doi:10.1158/1538-7445.AM2013-4371
Abstract 4372: Evaluating TBK1 as a cancer therapeutic target in cancer cell lines with activated IRF3.
Authors: Muvaffak Asli , Pan Qi , Middleton Richard , Morris Erick , Fernandez Rafael , Lim Jonwong , Dolinski Brian , Nguyen Thi T. , Strack Peter , Yan Haiyan , Chung Rossana , Wu Stephen , Zhang Weiqun , Hulton Chris , Hirsch Heather , Nagashimo Kumiko , Wang Yan
Published: August 2014

TBK1 is a non-canonical IκB protein kinase that phosphorylates and activates downstream targets such as IRF3 and cRel and, reportedly mediates NFkB activation in cancer. We used both genetic and pharmacological approaches to evaluate TBK1 function in cancer cells. In oncogenic KRAS mutant lung cancer cell lines, the TBK1 knockdown did not correlate with reduction in a 2D viability assay. Using TBK1 small molecule inhibitors, we found that the inhibition of cell growth in 2D viability assays did not correlate with Kras dependency or RAS genetic signature. Since the pIRF3(S386) levels in lung lines were too low to assess TBK1 target engagement, we identified cell lines with high pIRF3(S386) levels. We discovered a subset of pancreatic and colorectal cell lines that have high constitutive pIRF3(S386) levels and this correlated with high levels of pTBK1(S172). TBK1 inhibitors were able to dose-dependently inhibit pIRF3S386 in pancreatic & colon lines. These observations indicate that pIRF3(S386) could be used as a biomarker for direct target engagement of TBK1 in cancer cell lines with activated TBK1 pathway. pTBK1(S172), a phosphorylation site within the TBK1 kinase domain activation loop, does not decrease upon inhibition with TBK1 inhibitors but, instead increases. This suggests stimulation of a feedback loop resulting in the activation of an upstream kinase(s) responsible for activating TBK1 in cancer cell lines. Although several pancreatic and colon cell lines were identified to have low μM sensitivity to TBK1 inhibitors, TBK1 shRNA knockdown did not impact proliferation in these cell lines even though pIRF3(S386) levels were reduced. Further studies using knockdown of the related IKKe homolog in cell lines with activated pathway are also being pursued to determine if IKKe knockdown alone or in combination with TBK1 knockdown would result in more substantial growth reduction. It is likely that inhibition of additional nodes in the NF-kB pathway and other pathways are needed to achieve robust anti-tumor efficacy.

Citation Format: Asli Muvaffak, Qi Pan, Richard Middleton, Erick Morris, Rafael Fernandez, Jonwong Lim, Brian Dolinski, Thi T. Nguyen, Peter Strack, Haiyan Yan, Rossana Chung, Stephen Wu, Weiqun Zhang, Chris Hulton, Heather Hirsch, Kumiko Nagashimo, Yan Wang. Evaluating TBK1 as a cancer therapeutic target in cancer cell lines with activated IRF3. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4372. doi:10.1158/1538-7445.AM2013-4372
Abstract 4375: Hrk mediates 2-methoxyestradiol-induced mitochondrial apoptotic signaling in prostate cancer cells.
Authors: Chang Inik , Majid Shahana , Saini Sharanjot , Zaman Mohd S. , Yamamura Soichiro , Chiyomaru Takeshi , Shahryari Varahram , Fukuhara Shinichiro , Deng Guoren , Dahiya Rajvir , Tanaka Yuichiro
Published: August 2014

Prostate cancer is one of the most prevalent cancers in males and ranks the second most common cause of cancer related deaths. 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, is a promising anticancer agent for various types of cancers. Although 2-ME has been shown to activate Jun N-terminal kinase (JNK) and mitochondrial dependent apoptotic signaling pathways, the underlying mechanisms including downstream effectors remain unclear. Here, we report that the human Bcl-2 homology 3 (BH3)-only protein harakiri (Hrk) is a critical effector of 2-ME-induced-JNK dependent apoptosis in prostate cancer cells. Hrk mRNA and protein are preferentially up-regulated by 2-ME, and Hrk induction is dependent on JNK activation of c-Jun. Hrk knockdown prevents 2-ME-mediated apoptosis by attenuating the decrease in mitochondrial membrane potential, subsequent cytochrome c (cyt c) release and caspase activation. Conversely, Hrk over-expression increases caspase-dependent prostate cancer cell apoptosis. Involvement of the pro-apoptotic protein Bak in this process suggested the possible interaction between Hrk and Bak. Thus, Hrk activation by 2-ME or its over-expression displaced Bak from the complex with anti-apoptotic protein Bcl-xL, while deletion of the Hrk BH3 domain abolished its interaction with Bcl-xL, reducing the pro-apoptotic function of Hrk. Finally, Hrk is also involved in the 2-ME-mediated reduction of X-linked inhibitor of apoptosis (XIAP) through Bak activation in prostate cancer cells. Together, our findings suggest that induction of the BH3-only protein Hrk is a critical step in 2-ME activation of the JNK-induced apoptotic pathway targeting mitochondria by liberating pro-apoptotic protein Bak.

Citation Format: Inik Chang, Shahana Majid, Sharanjot Saini, Mohd S. Zaman, Soichiro Yamamura, Takeshi Chiyomaru, Varahram Shahryari, Shinichiro Fukuhara, Guoren Deng, Rajvir Dahiya, Yuichiro Tanaka. Hrk mediates 2-methoxyestradiol-induced mitochondrial apoptotic signaling in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4375. doi:10.1158/1538-7445.AM2013-4375
Abstract 4379: Framing therapeutic opportunities in tumor-activated gametogenic programs.
Authors: Corcoran Kathleen , Taus Patrick , Sinnott Rebecca , Maxfield Kimberly , Wooten Joshua , Whitehurst Angelique
Published: August 2014

Tumors frequently activate genes whose expression is otherwise restricted to testes, and which can invoke a humoral immune response in cancer patients. Given their germline-biased expression pattern, these Cancer-Testes antigens (CT-antigens, CTAs) may afford the potential for an extraordinary therapeutic window, if they are required for tumorigenic behaviors. While the function of CTAs in normal and neoplastic settings remains obscure, mounting evidence suggests that specific CTAs can contribute to tumor cell autonomous behaviors. To generate a more comprehensive understanding of the functional roles of CTAs, we have employed an unbiased, multi-dimensional, loss of function discovery platform to systematically assess the contribution of CT-antigens to a range of tumorigenic signaling pathways and phenotypes. By employing a diverse panel of 11 tumor-derived cell lines to screen 120 CTAs, we find that these tumor-activated genes are required to support a number of the hallmarks of cancer. For example, we reveal multiple MAGE family members that mitigate response to mitotic stress and hypoxia. Additionally, we find that the SPANX family of CTAs contributes to β-catenin signaling. Finally, we reveal a role for GAGE proteins in supporting the viability of a range of tumor types. The diversity of neoplastic settings analyzed in our primary screen allowed for the identification of CTAs that support tumor cell fitness in multiple disease backgrounds. For example, we have uncovered CTAs that contribute to mitochondrial integrity in lung, breast, melanoma and sarcoma. Alternatively, we find that CTAs can display lineage selective dependencies, such as supporting TGFβ signaling in claudin-low breast tumors. Taken together, these observations support the hypothesis that CTAs are frequently integrated into the tumor cell regulatory environment to support cell division, survival and modulate stress responses by regulating key signaling pathways. Thus, CT-antigens present previously unrecognized tumor-specific vulnerabilities that may be exploited for therapeutic intervention.

Citation Format: Kathleen Corcoran, Patrick Taus, Rebecca Sinnott, Kimberly Maxfield, Joshua Wooten, Angelique Whitehurst. Framing therapeutic opportunities in tumor-activated gametogenic programs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4379. doi:10.1158/1538-7445.AM2013-4379
Abstract 438: Establishment of MDA-MB231-derived breast cancer cell lines possessing a higher metastatic activity and a resistance to radiation.
Authors: Hara Takamitsu , Iwadate Manabu , Shiratori Shuichi , Gonda Kenji , Shimura Tatsuo , Nakagami Yoshihiro , Shibata Masahiko , Waguri Satoshi , Takenoshita Seiichi
Published: August 2014

Background and purpose: Metastatic cancer tissues might have characteristics different from their original primary regions, which could affect strategies for radiotherapy. To investigate properties of metastatic cancers and their radiosensitivities, we established MDA-MB231-derived breast cancer cell lines, which were prone to cause metastasis when transplanted into mouse.

Methods: A breast cancer cell line, MDA-MB231 expressing pGL4.5/luciferase were injected into nude mice via left ventricle or mammary gland tissue to form metastatic lesions. The metastatic cells in lymph nodes, bone, and lung were detected by a bioluminescence imaging technique, which were excised, cultured, and transplanted into the next mice in the same way. Such transplantation procedures were repeated 3 times to establish organ-oriented metastasis-prone cell lines. The different characteristics of these cells were examined by morphological analysis, and proliferation, invasion, and migration assays, as well as radiation sensitivity test.

Result: There was no significant difference among cell lines in morphology and proliferative ability. All three metastasis-prone cell lines showed increased activity in migration and invasion compared to the parental cell line. Moreover, they showed lower sensitivities to radiation than the parental cell line, while no difference was found among the metastasis-prone cell lines.

Conclusion: We have established new cell lines that could be used for the study of metastatic breast cancers in relation to the radiotherapy.

Citation Format: Takamitsu Hara, Manabu Iwadate, Shuichi Shiratori, Kenji Gonda, Tatsuo Shimura, Yoshihiro Nakagami, Masahiko Shibata, Satoshi Waguri, Seiichi Takenoshita. Establishment of MDA-MB231-derived breast cancer cell lines possessing a higher metastatic activity and a resistance to radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 438. doi:10.1158/1538-7445.AM2013-438
Abstract 4384: Discovery of CD146/Latexin as a novel pathway suppressing breast tumor invasion.
Authors: Ouhtit Allal , Abd Elmageed Zakariya Y. , Fernando Augusta , Gaur Rajiv , Gupta Ishita , Shanmuganathan Somya , Al-Riyami Hamad , Raj Madhwa HG
Published: August 2014

CD146, also known as melanoma cell adhesion molecule or MUC18, was initially identified as a promoter of melanoma as well as prostate cancer progression and metastasis. Interestingly, however, CD146 appears to be a breast cancer (BC) tumor suppressor. The molecular mechanism of CD146 in cancer are still nascent and controversial. To address this discrepancy, we applied microarray gene expression profiling analysis, using a tetracycline (tet On)-inducible system of CD146 in the MDA-MB-231 BC cell line, and identified latexin (LXN) as a potential CD146-downstream signaling transcriptional target. LXN showed a 7 fold increase, which was confirmed in vitro by both time-course western blotting and RT-PCR analyses. In addition, NF-kappaB was identified as the molecular link between CD146 induction and LXN transactivation.

More interestingly, we established the CD146-tet On inducible system in mouse BC xenograft model, and demonstrated that in vivo induction of CD146-inhibited breast tumor growth. Furthermore, breast tumor tissues from both Omani patients and patients from New Orleans area in Louisiana, were examined by immunohistochemistry for the expression of CD146 and LXN. Strikingly, the results revealed that the expression levels of both CD146 and its downstream target LXN showed parallel inhibition patterns in BC progression, with high expression in both normal and benign but low or no expression in malignant and metastatic breast tumor tissues.

This study is the first to uncover a molecular link and provide evidence for a role of CD146/LXN-signaling in suppressing BC progression. Thus, reactivation of LXN through pharmacological manipulation holds particular promise for guiding the development of novel targeted-therapeutic strategies for BC.

Citation Format: Allal Ouhtit, Zakariya Y. Abd Elmageed, Augusta Fernando, Rajiv Gaur, Ishita Gupta, Somya Shanmuganathan, Hamad Al-Riyami, Madhwa HG Raj. Discovery of CD146/Latexin as a novel pathway suppressing breast tumor invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4384. doi:10.1158/1538-7445.AM2013-4384
Abstract 440: The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene.
Authors: Lo Chia-Chien , Chou Yen-Ting , Chiu Su-Jun , Hwang Jeng-Jung , Lee Yi-Jang
Published: August 2014

The sublethal dose radiation may induce cellular senescence and promote secondary maligancy during radiotherapy. However, the molecular mechanisms are unclear. It has been reported that a variety of early response oncogenes can be up-regulated by the low dose radiation. Because oncogene-induced senescence (OIS) has been considered important for tumor development, we are interested in investigating whether the sublethal dose radiation also raises OIS to affect the malignancy. To this end, human diploid fibroblasts WI-38 cells and human non-small lung cancer H1299 cells were exposed to less than 2Gy of X-rays delivered by the RS2000 X-rays machine. Both cell types exhibited senescent phenotypes within a dose-dependent manner using the senescence-associated β-galactosidase (SA-β-gal) staining. Subsequently, we investigated the expression of c-Myc and c-Ras oncogenes, and the results showed that sublethal dose radiation could induce c-Myc, but not c-Ras, in both transcriptional and translational levels. Additionally, up-regulated c-Myc exhibited transcriptional activity as demonstrated by the promoter-based reporter gene assys. Although the sublethal dose radiation caused cellular senescence and growth delay in exposed cells, the collected culture medium could increase the motility of non-irradiated H1299 cancer cells using the wound healing assay. Interestingly, the culture medium obtained from irradiated H1299 cancer cells caused higher motility in non-irradiated cells than that from irradiated WI-38 fibroblasts. Furthermore, the sublethal dose radiation induced senescent phenotype was suppressed in cells treated with siRNA against c-Myc. Meanwhile, the motility of non-irradiated cells was not increased by the cultured medium obtained from irradiated c-Myc silenced cells. Taken together, the current data suggest that the sublethal dose radiation causes a c-Myc oncogene induced senescence, and this event may promote the bystandard effects that influence the motility of non-irradiated cancer cells.

Citation Format: Chia-Chien Lo, Yen-Ting Chou, Su-Jun Chiu, Jeng-Jung Hwang, Yi-Jang Lee. The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 440. doi:10.1158/1538-7445.AM2013-440

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 4401: UCHL1 as a potential therapeutic target in uterine papillary serous carcinoma.
Authors: Kwan Suet Ying , Wong Kwong-Kwok , Schmandt Rosemarie E. , Lu Karen H.
Published: August 2014

Uterine papillary serous carcinoma (UPSC) comprises less than 10% of all endometrial cancers but accounts for a disproportionately high number of deaths. Due to its rarity, few prospective studies have been conducted which focus on this aggressive subtype, and it has been difficult to establish optimal treatment strategies. With poor survival rates and high recurrence rates, the identification of novel therapeutic targets would aid in the management of UPSCs. RNAseq data from The Cancer Genome Atlas (TCGA) was analyzed using Significant Analysis of Microarrays to identify genes that were differentially expressed between 81 cases of grade 2 endometrioid endometrial carcinoma (EEC) and 41 cases of UPSC. A gene of interest was then selected based on its subcellular location, correlation with survival and normal tissue specificity. The findings were validated by immunohistochemical staining of UPSC paraffin sections. All patient samples were obtained and stored in accordance with human subject research protocols approved by the Institutional Review Board. All paraffin sections were reviewed by a pathologist. By comparing the RNAseq profiles of UPSCs and EECs, we identified 242 genes that were differentially expressed (q<0.005). Among these, ubiquitin C-terminal hydrolase 1 (UCHL1) showed higher expression in UPSCs compared to EECs, with an increase of over 6-fold. UCHL1 was chosen for further studies as Kaplan-Meier analysis of all endometrial cases in the TCGA database revealed that UCHL1 expression also correlated significantly with poorer disease-free survival (p<0.05). Preliminary immunohistochemical studies also show higher expression of UCHL1 in UPSCs than in ECCs. Interestingly, contrast analysis of TCGA data for breast cancer indicates that basal-like breast tumors also show significantly higher UCHL1 expression compared to the other breast cancer subtypes (p<0.005). Both basal-like breast cancer and UPSC have a high rate of p53 mutation (80% and 90%); this shared upregulation of UCHL1 suggests that a possible mechanism by which UCHL1 contributes to pathogenesis is through stabilization of mutant p53. In conclusion, our data identifies UCHL1 as a gene that is upregulated in UPSCs and is associated with poor disease-free survival. UCHL1 is a possible novel therapeutic target for the management of UPSCs. Further studies are needed to elucidate the underlying mechanisms in which it contributes to tumorigenesis.

Citation Format: Suet Ying Kwan, Kwong-Kwok Wong, Rosemarie E. Schmandt, Karen H. Lu. UCHL1 as a potential therapeutic target in uterine papillary serous carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4401. doi:10.1158/1538-7445.AM2013-4401
Abstract 4405: RNases: Approaching RNA as a target for cancer therapy.
Authors: Strong Laura E. , Kink John , Mei Baigen , Shahan Mark , Raines Ronald T.
Published: August 2014

Ribonucleases (RNases) are a unique class of anti-cancer drugs that target the RNA in cancer cells without the complications of delivering RNA. Efficacious RNases have diminished binding to the naturally occurring RNase inhibitor protein in the cytosol of cells. Since human RNase I binds tightly to the inhibitor, changes to the amino acid residues in the contact region were made and resulted in the lead candidate referred to as QBI-139. This variant maintains 95% sequence identity with the wild type human RNase 1.

Broad efficacy and a strong therapeutic window were seen across multiple tumor types in xenograft models and QBI-139 was advanced to an ongoing Phase I clinical trial. For the Investigational New Drug application package, rat and dog toxicology studies were used to aid in selection of the starting dose for humans. Exposure levels (pharmacokinetics, PK) are an important factor in evaluating whether toxicity and efficacy will be comparable across the species. A complicating issue in the evaluation of the results is the range of routes of administration across species, specifically intraperitoneal injection (mice), intravenous injection (rat and dog) and intravenous infusion (human). One parameter that is consistent is rapid clearance, which is likely due to the small size of QBI-139 (15 kiloDaltons). With preliminary results from the early stage clinical trial, the exposure levels across the experiments will be compared. The results demonstrate that the exposure profiles in the clinic where the route of administration is intravenous infusion, is most similar to that in the mouse efficacy models, which is intraperitoneal injection.

A human RNase variant, called QBI-139 provides an innovative drug development approach to targeting RNA. In model systems, QBI-139 has shown broad in vivo anti-tumor activity alone and in combination with standard of care therapies and a strong tolerability profile. The knowledge gained about the impact of PK on therapeutic effect will be valuable as the method of administration is selected and confirmed in the next stage of clinical trials.

Citation Format: Laura E. Strong, John Kink, Baigen Mei, Mark Shahan, Ronald T. Raines. RNases: Approaching RNA as a target for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4405. doi:10.1158/1538-7445.AM2013-4405
Abstract 4407: Structural and biochemical analysis of ERBB4 mutations in cancer.
Authors: Kurppa Kari , Denessiouk Konstantin , Johnson Mark S. , Elenius Klaus
Published: August 2014

Genes encoding the ErbB receptor tyrosine kinases (EGFR/ERBB1, ERBB2, ERBB3, and ERBB4) are key regulators of cellular proliferation, survival, and differentiation, and thus represent potent proto-oncogenes. In particular, mutations or copy number variations of EGFR or ERBB2 are present in human malignancies and serve as biomarkers for targeted therapies. Recently, somatic mutations of the ERBB4 gene have been identified in various cancer types, such as non-small cell lung cancer and melanoma, indicating a role for mutated ERBB4 as a potential drug target. However, the structural and functional consequences of the ERBB4 mutations remain to be fully elucidated.

We used both a structural as well as a biochemical approach to address the functional effects the ERBB4 mutations for ErbB4 signaling. The structural effects of the mutations were assessed in both active and inactive ErbB4 conformation in silico. The biochemical effects of the mutations on ErbB4 signaling were analyzed in vitro using assays measuring the kinase activity of ErbB4 as well as activation of downstream signaling pathways.

Although all structural subdomains of ERBB4 have been reported to be affected by somatic mutations, our analyses indicated that the mutations cluster to regions important for activation of the receptor, such as the dimerization interface and the kinase domain active site. Some of the mutations were structurally non-hazardous and probably represent passenger mutations. Consistently, the biochemical analyses indicated for the presence of both activating and functionally non-significant mutations.

These observations suggest for the presence of both driver as well as passenger mutations of ERBB4 in human cancer.

Citation Format: Kari Kurppa, Konstantin Denessiouk, Mark S. Johnson, Klaus Elenius. Structural and biochemical analysis of ERBB4 mutations in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4407. doi:10.1158/1538-7445.AM2013-4407
Abstract 4412: S1 and gossypol induce NOXA through increased reactive oxygen species or cytoplasmic calcium and sensitize to ABT-737.
Authors: Soderquist Ryan S. , Bates Darcy J. P. , Eastman Alan
Published: August 2014

The upregulation of anti-apoptotic BCL2 family members enhances the survival of cancer cells, such as in chronic lymphocytic leukemia (CLL). A class of inhibitors (termed BH3 mimetics) has been developed to inhibit BCL2 proteins and selectively kill cancer cells. These inhibitors function by occupying the groove through which BCL2 proteins interact with their binding partners. ABT-737 is a high affinity inhibitor of BCL2 and BCLXL, and navitoclax, which has a similar binding profile, has shown efficacy in clinical trials. However, resistance occurs when the cancer cells rely on additional BCL2 members, such as MCL1 or BFL1, which are not inhibited by ABT-737. Using leukemia cell lines, we compared several BH3 mimetics reported to inhibit multiple BCL2-family proteins (including MCL1 and BFL1), with the goal of enhancing the efficacy of ABT-737. We found that none of these compounds functioned as true BH3 mimetics. Instead, the compounds induced ER stress and upregulated the pro-apoptotic protein NOXA. Furthermore, two of these compounds, S1 and gossypol, sensitized leukemia cells to ABT-737 in a NOXA-dependent manner. NOXA induced by S1 or gossypol is dependent on increased reactive oxygen species or increased cytoplasmic calcium, respectively. We also tested these drug combinations in CLL cells ex vivo. S1 and gossypol combined with ABT-737 to selectively kill CLL cells but not normal lymphocytes. Interaction between CLL cells and stroma in patients promotes resistance to BCL2 inhibitors through the upregulation of MCL1 and BFL1. Co-incubation of CLL cells ex vivo with CD40 ligand-expressing stroma mimics the resistance to BCL2 inhibitors seen in patients. Importantly, this resistance was overcome when S1 or gossypol was combined with ABT-737. Our results show two novel mechanisms for upregulating NOXA and sensitizing leukemia cells to ABT-737. These combinations also kill CLL cells exposed to stroma, and may therefore target residual CLL cells in vivo, thus improving clinical outcome.

Citation Format: Ryan S. Soderquist, Darcy J. P. Bates, Alan Eastman. S1 and gossypol induce NOXA through increased reactive oxygen species or cytoplasmic calcium and sensitize to ABT-737. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4412. doi:10.1158/1538-7445.AM2013-4412
Abstract 4416: A reporter mouse to measure drug myelotoxicity in time.
Authors: Rizzi Nicoletta , Manni Isabella , Mancini Laura , Roncoroni Chiara , Brunialti Electra , Oliva Paolo , Gentileschi Maria Pia , Di Giovine Stefano , Pesenti Enrico A. , Piaggio Giulia , Maggi Adriana , Ciana Paolo
Published: August 2014

Myelotoxicity is the well known side effect of most anti-cancer treatments, thus the study of the myelosuppressive effect of a given treatment is tested routinely during the preclinical phase of investigation. Current ethodologies require the use of a very large number of animals to measure the effect of the investigated compound in time and the study is generally carried out by bone marrow dissection and subsequent analysis with a myeloid clonogenic assay. The goal of our study was to apply non invasive imaging technologies to generate a novel, simple and robust assay to test the state of proliferation of bone marrow reducing the number of animals to be sacrificed and increasing the power of the analysis thanks t the possibility to follow in time, in the same animal the effect of the treatment. To this aim, we have utilized a trangenic mouse, the repTOP™mitoIRE reporter mouse recently developed to image proliferative events in vivo (Goeman et al 2012, Oliva et al 2012). This transgenic mouse carries a luciferase reporter gene under the transcriptional control of a minimal human cyclin B2 promoter.Our in vitro and in vivo results show that the luciferase expression and photon emission in the different body regions of the repTOP™mitoIRE is strictly proportional to the proliferative state of the cells in the tissues examined. Our results demonstrate that the extent of bone marrow proliferation can be measured by in vivo imaging photon emission of femur and sternum in each mouse prior the treatment and then monitored during the longitudinal study.

The repTOP™mitoIRE mouse model, was successfully applied to the study of the differential immunosuppressive potential of several well known anti-neoplastic treatments demonstrating the validity of the novel test over currently available methodologies: we measured the differential effects on immunosuppression and on regeneration of 5-fluorouracil, docetaxel, bortezomid, temozolomide and radiotherapy in dose-response experiments. The results led us to conclude that the imaging-based test here proposed is reproducible and provides a reliable measure of bone marrow cell proliferation in time and to propose it as a routine test in for the pre-clinical of novel anti-neoplastic compounds.

Citation Format: Nicoletta Rizzi, Isabella Manni, Laura Mancini, Chiara Roncoroni, Electra Brunialti, Paolo Oliva, Maria Pia Gentileschi, Stefano Di Giovine, Enrico A. Pesenti, Giulia Piaggio, Adriana Maggi, Paolo Ciana. A reporter mouse to measure drug myelotoxicity in time. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4416. doi:10.1158/1538-7445.AM2013-4416