bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2465: p53-dependent and independent functions of Mieap
Authors: Nakamura Yasuyuki , Kamino Hiroki , Saito Yuri , Sano Hitoya , Arakawa Hirofumi
Published: May 2015

Mieap, a p53-inducible protein, plays a pivotal role in mitochondrial quality control (MQC) by repairing or eliminating unhealthy mitochondria1-4. To investigate physiological significance of the Mieap-regulated MQC, we generated and analyzed the Mieap-KO mice. (1) p53-dependent: Hypoxia induced Mieap expression and accumulation of lysosomal proteins within mitochondria in WT MEFs, but not Mieap-/- and p53-/- MEFs. Mieap-/- and p53-/- MEFs exhibited accumulation of oxidized mitochondrial proteins and increased ROS generation by unhealthy mitochondria under hypoxia, which enhanced cell migration and invasion. (2) p53-dependent: Mieap+/- / ApcMIN/+ mice displayed a shortening of the lifetime, compared to the ApcMIN/+ mice (average life-span: Mieap+/- / ApcMIN/+ mice vs ApcMIN/+ mice = 19.1 weeks vs 24.4 weeks), suggesting that Mieap deficiency may affect intestinal tumorigenesis. (3) p53-independent: Mieap was highly expressed in the midpiece, where the sperm mitochondria are localized, of the sperm in WT mice. This Mieap expression was independent on p53. Mieap-/- mice are fertile in vivo. However, in vitro fertilization of Mieap-/- sperm was severely impaired because of a failure of sperm motility. The oxidized proteins were dramatically accumulated in the midpiece of Mieap-/- sperm. These results, taken together, suggest that Mieap plays a crucial role in protection of the cell against oxidative stresses probably through its MQC function. This may also largely contribute to p53 tumor suppression.

1. Miyamoto Y et al. PLoS ONE 6: e16054, 2011

2. Kitamura N et al. PLoS ONE 6: e16060, 2011

3. Nakamura Y et al. PLoS ONE 7: e30767, 2012

4. Miyamoto T et al. Sci Rep 2: 379, 2012

Citation Format: Yasuyuki Nakamura, Hiroki Kamino, Yuri Saito, Hitoya Sano, Hirofumi Arakawa. p53-dependent and independent functions of Mieap. [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 2465. doi:10.1158/1538-7445.AM2014-2465
Abstract 2468: A p53 and microRNA-mediated pathway regulates mesenchymal stem cell differentiation
Authors: He Yunlong , Jiang Shunlin , Dubois Wendy , Ren Ling , Khanna Chand , Huang Jing
Published: May 2015

The tumor suppressor p53 plays important roles in bone density regulation and osteosarcoma suppression. However, the relationship between p53-mediated bone density regulation and osteosarcoma suppression is unclear mainly because the underlying molecular and cellular mechanisms are elusive. Here, we reported that p53 controls differentiation of mouse mesenchymal stem cells (mMSCs) in a cell cycle and apoptosis independent manner. The loss of p53 predisposes mMSCs towards osteogenic lineage but not adipogenic and chondrogenic lineages. This pro-osteogenic effect caused by p53 loss is associated with the up-regulation of Runt related transcription factor 2, a factor required for the osteogenic differentiation of mMSCs. Without extrinstic stress, studies show that p53 does not directly repress the transcription of Runt related transcription factor 2. Instead, it indirectly represses Runt related transcription factor 2 through activating microRNAs (miRs), which suppress Runt related transcription factor 2 at a post-transcriptional level. In osteosarcoma cells, the loss of p53 correlates with high levels of Runt related transcription factor 2 and the oncogene addiction of these cells to Runt related transcription factor 2. Together, our results discover a p53 and miR-mediated pathway regulating the differentiation of MSCs and this pathway is dysregulated in osteosarcoma cells. This lineage controlling role links the role of p53 in the osteogenic differentiation of MSC to its suppressive functions in osteosarcoma.

Citation Format: Yunlong He, Shunlin Jiang, Wendy Dubois, Ling Ren, Chand Khanna, Jing Huang. A p53 and microRNA-mediated pathway regulates mesenchymal stem cell differentiation. [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 2468. doi:10.1158/1538-7445.AM2014-2468
Abstract 2470: Cd177, a novel metastasis suppressor of breast cancer
Authors: Xie Qing , Borcherding Nicholas , Kolb Ryan , Zhang Weizhou
Published: May 2015

Mammary glands contain luminal and basal epithelial cells. We recently found that both luminal and basal epithelial cells can initiate malignant mammary tumors with forced ErbB2/Her2/c-Neu expression (referred to as luminal and basal tumor-initiating cells, respectively). Basal tumor-initiating cell (TIC)-derived tumors had a greater incidence and larger volume than luminal TIC-derived tumors. Transcriptomic analysis was performed to evaluate the underlying transcriptional difference between ErbB2-driven luminal and basal TIC-derived tumors. CD177 was found to be downregulated within basal TIC-derived tumors. CD177 is an important membrane glycoprotein on neutrophils and serves as a marker for myeloproliferative diseases. The role of CD177 in solid tumors is unknown. Our initial analysis revealed that loss of CD177 expression is linked to higher metastasis incidence and shorter survival in breast cancer patients, but not other diagnostic markers like estrogen-, progesterone-, or HER2-receptors. Silencing CD177 in 4T1 cells, a mouse mammary tumor cell line, led to a significant increase for colonic growth in soft agar and pulmonary metastasis in mice. Overexpressing CD177 in MCF7 cells, a human luminal mammary tumor cell line, resulted in a significant decrease for colonic growth in soft agar. We also found that genetic deletion of CD177 in female mice results in significantly increased Lineage-CD24+CD49fhi basal epithelial cells, the major tumor-initiating cells for ErbB2-induced breast cancer. Collectively, we identified a novel potential metastasis-suppressor for human breast cancer with important prognostic value. Future studies will involve in the mechanistic understanding of how CD177 regulates metastasis of breast cancer.

Citation Format: Qing Xie, Nicholas Borcherding, Ryan Kolb, Weizhou Zhang. Cd177, a novel metastasis suppressor of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2014-2470
Abstract 2471: Direct down-regulation of eEF1A2 by Tumor suppressor p16INK4a inhibits cancer cell growth
Authors: Lee Mee-Hyun , Choi Bu Young , Cho Yong-Yeon , Kim Myoung O. , Kim Sung-Hyun , Lee Cheol-Jung , Song Ji Hong , Bode Ann M. , Dong Zigang , Surh Yong-Joon
Published: May 2015

The tumor suppressor protein p16INK4a is a member of the INK4 family of cyclin-dependent kinase (Cdk) inhibitors, which are involved in the regulation of the eukaryotic cell cycle. However, the mechanisms underlying the anti-proliferative effects of p16INK4a have not been fully elucidated. Using yeast two-hybrid screening, we identified the eukaryotic elongation factor (eEF)1A2 as a novel interacting partner of p16INK4a. eEF1A2 is thought to function as an oncogene in cancers. The p16INK4a protein interacted with all but the D2 (250-327 aa) domain of eEF1A2. Computational docking study predicted that D24/D131 residues of p16INK4a interacted with eEF1A2 and it was confirmed by pull-down assay with mutant p16INK4a (D24A/D131E). Ectopic expression of p16INK4a decreased the expression of eEF1A2 and inhibited cancer cell growth. Furthermore, suppression of protein synthesis by expression of p16INK4a ex vivo was verified by luciferase reporter activity. Microinjection of p16INK4a mRNA into the cytoplasm of Xenopus embryos suppressed the luciferase mRNA translation, whereas the combination of p16INK4a and morpholino-eEF1A2 resulted in a further reduction in translational activity. We conclude that the interaction of p16INK4a with eEF1A2, and subsequent downregulation of the expression and function of eEF1A2 is a novel mechanism explaining the anti-proliferative effects of p16INK4a.

Citation Format: Mee-Hyun Lee, Bu Young Choi, Yong-Yeon Cho, Myoung Ok Kim, Sung-Hyun Kim, Cheol-Jung Lee, Ji Hong Song, Ann M. Bode, Zigang Dong, Yong-Joon Surh. Direct down-regulation of eEF1A2 by Tumor suppressor p16INK4a inhibits cancer cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2014-2471
Abstract 2472: Acute colitis and colitis-associated cancer are exacerbated in mice deficient N-deacetylase/N-sulfotransferase-4
Authors: Jao Tzu-Ming , Tsai Ming-Hong , Huang Chi-Yen , Tzeng Sheng-Tai , Lee Jing-Xing , Lo Wei-Chen , Chang Che-Wei , Yang Ya-Chien
Published: May 2015

NDST4 is one member of N-deacetylase/N-sulfotransferase (heparan glucosaminyl) (NDST) family, which are responsible for heparan sulfate (HS) biosynthesis on a core protein to form heparan sulfate proteoglycans (HSPGs). The HS chains of HSPGs bind to and regulate their functions of various growth factors, cytokines and chemokines. HSPGs ubiquitously reside on cell surface, inside the cell, and in the extracellular matrix. Importantly, the content and distribution of HSPGs are altered during tumorigenesis. In the study, 30 (57.7%) of 52 primary colorectal carcinomas showed a dramatic reduction in NDST4 RNA transcripts, and genetic loss of NDST4 was significantly associated with poor survival of patients with colorectal cancer. We further generated an Ndst4-knockout mouse strain, which could develop and reproduce normally. Notably, using the dextran sodium sulfate mouse model of acute colitis, Ndst4 deficiency resulted in a significantly higher disease activity index and obviously hyperemic appearance in the cecum. In the development of colitis-associated cancer, the size of colonic tumors induced by azoxymethane/dextran sodium sulfate was significantly increased in Ndst4-deficient mice compared with wild-type mice. Taken together, loss-of-function of NDST4 might impair the modification of HS chains of specific HSPGs leading to tumor-promoting inflammation in the colon. The full spectrum of signaling pathways contributed by NDST4 to colitis and tumor progression remains to be elucidated.

Citation Format: Tzu-Ming Jao, Ming-Hong Tsai, Chi-Yen Huang, Sheng-Tai Tzeng, Jing-Xing Lee, Wei-Chen Lo, Che-Wei Chang, Ya-Chien Yang. Acute colitis and colitis-associated cancer are exacerbated in mice deficient N-deacetylase/N-sulfotransferase-4. [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 2472. doi:10.1158/1538-7445.AM2014-2472
Abstract 248: Vitamin D deficiency promotes hepatocellular carcinoma tumor growth in TGF-β impaired mice by Smad3 heterozygous deletion
Authors: Muñoz Nina M. , Katz Lior H. , Machida Keigo , Tsukamoto Hidekazu , Shetty Kirty , He Aiwu R. , Johnson Lynt B. , Rashid Asif , Kim Sang Bae , Lee Ju-Seog , Mishra Lopa
Published: May 2015

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers, some of which are characterized by inactivation of TGF-β signaling. Recently, inactivation of TGF-β signaling has been associated with development of HCC. We examined the potential role of VD in HCC chemoprevention in the context of TGF-β inactivation in a mouse model. Methods: Wild type, Sptbn1+/- and Smad3+/- male mice were treated with 50 mg/kg of the chemical carcinogen diethylnitrosamine (DEN) via i.p. injection at 14 days of age. At 8 months, the animals were changed from regular chow (2,200 IU/kg VD) to one of two diets: lower than normal VD (200 IU/kg) or high VD (10,000 IU/kg). At one year of age, the livers were harvested for tissue analysis. Several metrics were assessed including expression levels of 164 proteins from liver tumors by Reverse Phase Protein Array (RPPA). Results: Twenty three mice were treated with lower than normal dose VD while 26 received high dose VD. We did not find significant differences in blood calcium levels in animals on either diet. As expected, high VD intervention after 8 months of DEN injection did not affect the total number of tumors the mice developed; however, the lower than normal VD regimen promoted tumor growth in the context of Smad3 disruption (liver-to-body weight ratio 18.37% in Smad3+/- mice treated with low dose VD vs. 6.25% in Smad3+/- animals treated with high dose, p=0.028). Furthermore, RPPA data revealed that the lower than normal VD intake in DEN-treated Smad3 heterozygous null mice results in repression of tumor suppressing genes such as PDCD4 and concomitant up-regulation of tumor promoting genes like Stat5A, Bcl2-XL, PDGFRB and PEA15. Conclusions: Remarkably, lower than normal VD in the context of Smad3 disruption promotes tumor growth possibly through repression of tumor suppressing genes such as PDCD4 and up-regulation of oncogenes like Stat5A, Bcl2-XL, PDGFRB and PEA15. These results suggest that VD treatment strategies could potentially be significant for chemoprevention and treatment of HCC in the context of inactivation of TGF-β signaling in patient populations with underlying deficiency of VD.

Citation Format: Nina M. Muñoz, Lior H. Katz, Keigo Machida, Hidekazu Tsukamoto, Kirty Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Sang Bae Kim, Ju-Seog Lee, Lopa Mishra. Vitamin D deficiency promotes hepatocellular carcinoma tumor growth in TGF-β impaired mice by Smad3 heterozygous deletion. [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 248. doi:10.1158/1538-7445.AM2014-248
Abstract 2528: Optimization of small molecules targeting BMI1 protein expression. Part 2. Improved potency, oral bioavailability, and in vivo efficacy of amino-pyrazines and amino-pyridines
Authors: Baiazitov Ramil , Sydorenko Nadiya , Ren Hongyu , Du Wu , Paget Steve , Wilde Richard , Liu Ronggang , Lee Chang-Sun , Cao Liangxian , Davis Thomas W. , Almstead Neil G. , Moon Young-Choon
Published: May 2015

Earlier, in our efforts to identify molecules that selectively deplete tumor stem cells, we discovered and elaborated on a series of amino-thiazoles. These first-in-class small molecule inhibitors reduce the activity and levels of BMI1 protein in vitro and in vivo (see the preceding poster).

Through our medicinal chemistry efforts, we developed a second generation of molecules that reduce levels of BMI1. In this new scaffold, the central thiazole ring has been replaced with a pyrazine or pyridine ring. There is a clear relationship between the structure of the central heterocycle and the ability of the compound to decrease the level of BMI1 protein.

The most beneficial modification on the right wing of the molecule is the introduction of benzimidazole moiety. As a result, analogs with markedly improved oral bioavailability have been identified.

The structure activity relationship on the left ring parallels the SAR observed with the amino-thiazole scaffold.

The most successful analogs in this series can be dosed orally and demonstrate dose-dependent efficacy in mouse xenograft models including the HT1080 and L1210 cell lines.

Citation Format: Ramil Baiazitov, Nadiya Sydorenko, Hongyu Ren, Wu Du, Steve Paget, Richard Wilde, Ronggang Liu, Chang-Sun Lee, Liangxian Cao, Thomas W. Davis, Neil G. Almstead, Young-Choon Moon. Optimization of small molecules targeting BMI1 protein expression. Part 2. Improved potency, oral bioavailability, and in vivo efficacy of amino-pyrazines and amino-pyridines. [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 2528. doi:10.1158/1538-7445.AM2014-2528
Abstract 255: Addition of biologic therapy to traditional disease-modifying anti-inflammatory drugs and the risk of cancer in patients with rheumatoid arthritis: a nationwide cohort study
Authors: Chen Yi-Ju , Wu Chun-Ying , Chen Der-Yuan , Shen Jui-Lung , Ho Hsiu J. , Liu Han-Nan , Chang Yun-Ting , Kuo Ken-Nan
Published: May 2015

Background: Rheumatoid arthritis (RA) is associated with certain types of cancer. The add-on effect of biologics on cancer occurrence in long-term RA remains unknown. We aimed to investigate the relative risk of cancer development among RA patients combining biologics with traditional disease-modifying anti-rheumatic drugs (DMARDs).

Methods: We conducted a nationwide cohort study between March 2003 and December 2010 using the Taiwan National Health Insurance Research Database (NHIRD). A total of 42,263 patients with RA who received long term DMARDs were identified. The risk of newly diagnosed cancer was compared between patients taking biologics in addition to DMARDs (biologics cohort, N=4,119) and four-fold propensity score-matched control patients taking nonbiologic DMARDs only (biologics-naive cohort, N=16,476). Cumulative incidences and hazard ratios (HR) were calculated after adjusting for competing mortality. Multivariate analyses were performed using Cox proportional hazards model.

Results: The incidence rates of cancer among biologics and biologics-naive cohorts were 2.09 (95% confidence interval [CI] 1.31-2.86) and 3.25 (95% CI 2.76-3.74) per 1000 person-years, respectively (p=0.03). On modified Cox proportional hazards analysis, the risk of cancer was significantly reduced in subjects in biologics cohort (adjusted HR 0.61, 95% CI 0.40-0.94, p=0.025) in comparison with subjects in biologics-naive cohort, after adjusting for age, gender, disease duration, major co-morbidities, and prior use of DMARDs and corticosteroids. The effect of biologics was consistent across all multivariate stratified analyses; and the association between biologics use and cancer risk was independent of dosage of concomitant use of non-biological DMARDs.

Conclusion: RA patients taking biologics plus long term traditional DMARDs therapy are associated with a lower risk of cancer when compared with RA patients taking traditional DMARDs alone.

Citation Format: Yi-Ju Chen, Chun-Ying Wu, Der-Yuan Chen, Jui-Lung Shen, Hsiu J. Ho, Han-Nan Liu, Yun-Ting Chang, Ken-Nan Kuo. Addition of biologic therapy to traditional disease-modifying anti-inflammatory drugs and the risk of cancer in patients with rheumatoid arthritis: a nationwide cohort study. [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 255. doi:10.1158/1538-7445.AM2014-255
Abstract 2550: Translational study on circulating markers in advanced melanoma patients undergoing Dacarbazine and Bevacizumab treatment
Authors: Ferrucci Pier Francesco , Martinoli Chiara , Cocorocchio Emilia , Anichini Andrea , Gandini Sara
Published: May 2015

Purpose:

Chemotherapy (CT) with Dacarbazine (DTIC) in association with an anti-VEGF treatment could be an intriguing exploiting path, since metastatic melanoma (MM) in vitro models showed VEGF as a possible target, due to its transcription up-regulation by DTIC. The purpose of this study was to indentify modulated circulating factors which may be used as specific biomarkers in patients (pts) affected by MM undergoing DTIC plus Bevacizumab (B = Avastin) combination and to test its tolerability and efficacy.

Patients and Methods:

Between July 2006-September 2009, 40 untreated MM pts underwent CT with DTIC (800mg/mq q4w) + B (10mg/kg q2w). Clinical benefit, response duration, safety and feasibility were analyzed. Moreover, 13 pts participating to the clinical study agreed to provide blood for translational research. The blood samples for this study were drawn at the same time points as for regular follow-up tests and were evaluated for the following several soluble factors: vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-R1, VEGF-R2, angiopoietin (Ang)-2, E-cadherin, E-selectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, matrix metalloproteinase (MMP)-2 and -9, tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Correlations with pts clinical outcome were performed as well.

Results:

36/40 pts were evaluated at the analysis-time. Median age was 57yr (male 60yr; female 47yr) and 23 were male. Three pts (8.3%) experienced a complete remission (CR), 3 pts (8.3%) developed partial response (PR), 13 (36.1%) stable disease (SD); 17 (47.2%) progressive disease (PD). Clinical benefit (CR+PR+SD) was 52.7% and treatment was well tolerated. Time to progression (months) was 10.9, respectively 22.6 for PR pts, 8.2 for SD pts and 2 for PD pts. Median-survival in the whole group was 16.5 months (PR pts 22.6; SD pts 17; PD pts 4.23). DTIC/B treatment modulates not only VEGF-A, but also VEGF-C, Ang-2 VEGF-R1 and VEGF-R2. Among soluble adhesion molecules, we observed an increase of VCAM-1 in non responder pts.

Conclusions:

Long-lasting responses obtained in both SD and CR/PR pts and mild side effects make DTIC/B a promising combination in advanced melanoma. The companion translational study partially clarified these clinical results by molecularly characterizing and profiling responders vs non-responders pts.

Citation Format: Pier Francesco Ferrucci, Chiara Martinoli, Emilia Cocorocchio, Andrea Anichini, Sara Gandini. Translational study on circulating markers in advanced melanoma patients undergoing Dacarbazine and Bevacizumab 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 2550. doi:10.1158/1538-7445.AM2014-2550
Abstract 2928: AZD6244 inhibits cisplatin induced ERK1/2 activation and potentiates its cytotoxic effects in NSCLC preclinical models
Authors: Kim Eun Young , Kim Arum , Byun Min Kwang , Kim Se Kyu , Kim Hyung Jung , Chang Joon , Ahn Chul Min , Chang Yoon Soo
Published: May 2015

Introduction; Lung cancer is still the leading cause of cancer death worldwide, indicating that there are urgent needs for elucidating the mechanisms of resistance and developments of strategies to overcome it. Cisplatin is still one of the most commonly used drugs for the treatment of advanced lung cancer and, however, the underlying mechanism of activation of MAPK/ERK1/2 after cisplatin administration is still complex and elusive. It is considered one of the main mechanisms mediating resistance to cisplatin based doublet chemotherapy. In this study we aimed to evaluate the effectiveness of combination of MAPK inhibitor, AZD6244, with cisplatin in NSCLC models. Materials and Methods; NSCLC cells harbouring oncogenic RAS mutation, NCI-H1299, -H2009, -H460, and A549 cells, and LSL K-Ras G12D mouse model was used. Protein expression was evaluated by immunoblotting and cytotoxicity was measured by FACS followed by annexin-V and PI staining. LSL Kras G12D mouse were inhaled 5X 107 PFU AdCre virus at 8 weeks after birth and, in 24 weeks after inhalation, were taken microCT, randomized into 4 groups, and then treated with AZD6244 (25mg/kg bid by oral gavage) and/or cisplatin (5 mg/kg weekly) for two weeks. Results; There was a dose dependent increase of pERK1/2, which is most prominent at 24 hr after cisplatin treatment. Expression of proapoptotic protein, BIM, was decreased whereas that of antiapoptotic proteins, BCL-2 and MCL-1, was increased with cisplatin treatment. Treatment of AZD6244 prevented phosphorylation mediated degradation of BIM after cisplatin treatment. AZD6244 itself did not show cytotoxic effects up to 10 uM whereas combination of 1 uM of AZD6244 potentiates cytotoxic effects of cisplatin. Combined treatment of AZD6244 with cisplatin showed significant tumor reduction in 2 weeks in LSL K-ras G12D mouse model when compared those treated with single agents or vehicle. Conclusion; Combination of AZD6244 with cisplatin potentiates cytotoxic effects and anti-tumor activities in NSCLC preclinical models. These findings suggest that clinical trials using combination with AZD6244 and cisplatin may be beneficial in advanced NSCLC patients.

Citation Format: Eun Young Kim, Arum Kim, Min Kwang Byun, Se Kyu Kim, Hyung Jung Kim, Joon Chang, Chul Min Ahn, Yoon Soo Chang. AZD6244 inhibits cisplatin induced ERK1/2 activation and potentiates its cytotoxic effects in NSCLC preclinical 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 2928. doi:10.1158/1538-7445.AM2014-2928
Abstract 2931: Cancer cells deficient in DNA mismatch repair (MMR) are selectively sensitive to inhibition of the DNA dependent protein kinase (DNA-PK)
Authors: Hewish Madeleine , Fontebasso Yari , Martin Sarah A. , Elliott Richard , Perks Kerry L. , Konde Asha , Bajrami Ilirjana , Weverwijk Antoinette Van , Cunningham David , Lord Christopher J. , Ashworth Alan
Published: May 2015

Many solid tumors, including a significant proportion of colorectal, endometrial, gastric and urothelial tract cancers, are functionally deficient in MMR. This results in an altered clinicopathological phenotype. With the objective of identifying potential MMR-deficient (dMMR) synthetic lethal vulnerabilities, we performed a large scale screen of drugs which included a library of kinase inhibitors.

Using a pair of isogenic colorectal cancer (CRC) cell lines, deficient and proficient for MLH1, we identified that exposure to an inhibitor of the catalytic subunit of DNA-PK (Ku-57788) was associated with a reduction in cell viability in MLH1-deficient cells at nanomolar concentrations. Validation studies demonstrated a ten-fold difference in sensitivity in clonogenic assays and a significant correlation between MMR status and Ku-57788 across a panel of cancer cell lines. Inhibition of expression of DNA-PK using RNA interference reproduced the dMMR selectivity. The dMMR phenotype was associated with a reduction in cellular proliferation, leading to apoptosis. Given the role of MMR in the repair of oxidative damage, we assayed levels of 8-oxo-dG, a marker of oxidatively damaged DNA, and observed increased levels in dMMR cells. Upregulation of the anti-oxidant response was observed on exposure to Ku-57788, whilst addition of antioxidants to culture media resulted in complete abrogation of dMMR selectivity. We assessed the effect of combining Ku-57788 with standard chemotherapeutics used in CRC and observed a supra-additive effect with irinotecan. In vivo, treatment of CD1 nude mice with a DNA-PK inhibitor was associated with a reduction in growth of MLH1-deficient cancer cell line xenografts with no effect observed in MLH1-proficiency.

We report for the first time that inhibition of DNA-PKcs using small molecule inhibitors, either as a single agent or in combination with chemotherapeutics, may be a potential therapeutic strategy for the treatment of cancers deficient in DNA MMR.

Citation Format: Madeleine Hewish, Yari Fontebasso, Sarah A. Martin, Richard Elliott, Kerry L. Perks, Asha Konde, Ilirjana Bajrami, Antoinette Van Weverwijk, David Cunningham, Christopher J. Lord, Alan Ashworth. Cancer cells deficient in DNA mismatch repair (MMR) are selectively sensitive to inhibition of the DNA dependent protein kinase (DNA-PK). [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 2931. doi:10.1158/1538-7445.AM2014-2931
Abstract 2949: YK-4-279 is a small molecule inhibitor of ETV1 and inhibits metastasis in a mouse model
Authors: Rahim Said , Justvig Sarah , Hong Sung-Hyeok , Tosso Perrer , Celik Haydar , Sayedigar-Kont Yasemin , Brown Milton , Morrissey Colm , Toretsky Jeffrey , Üren Aykut
Published: May 2015

The erythroblastosis virus E26 transforming sequences (ETS) family of transcription factors consists of a highly conserved group of genes that play important roles in cellular proliferation, differentiation, migration and invasion. Chromosomal translocations fusing ETS factors to promoters of androgen responsive genes have been found in a majority of prostate cancers, including the most clinically aggressive forms. ERG and ETV1 are the most commonly translocated ETS proteins. Over-expression of these proteins in prostate cancer cells results in a more invasive phenotype. The high prevalence of these rearrangements, and their biological significance represents a novel therapeutic target for the treatment of prostate cancer.

We recently demonstrated that the small molecule YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here, we present our findings in an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously implanted with fusion-positive LNCaP and fusion-negative PC-3 tumors. Animals were treated with YK-4-279 and its effect on tumor size, lung metastasis and survival were observed. YK-4-279 treatment resulted in decreased tumor size in the LNCaP cohort only. A reduction in tumor metastasis to the lungs was observed in compound treated LNCaP animals with comparable tumor sizes. YK-4-279 also increased survival in LNCaP mice. Expression of ETV1 target genes MMP7, FKBP10 and GLYATL2 was reduced as well. ETS fusion-negative PC-3 xenografts were unresponsive to the compound. YK-4-279 is a chiral molecule that exists as a racemic mixture of R and S enantiomers. As part of this study, we also established that (S) -YK-4-279 is the active enantiomer in prostate cancer cells. (S) -YK-4-279 binds to ETV1 with comparable kinetics as the racemic mixture and inhibits ETV1 activity. (R) -YK-4-279 does not demonstrate ETV1 binding or inhibition.

Our results demonstrate that YK-4-279 is a potent inhibitor of ETV1 and should be further evaluated for its clinical applications in prostate cancer.

Citation Format: Said Rahim, Sarah Justvig, Sung-Hyeok Hong, Perrer Tosso, Haydar Celik, Yasemin Sayedigar-Kont, Milton Brown, Colm Morrissey, Jeffrey Toretsky, Aykut Üren. YK-4-279 is a small molecule inhibitor of ETV1 and inhibits metastasis in a mouse model. [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 2949. doi:10.1158/1538-7445.AM2014-2949
Abstract 2951: Vitamin C is selectively toxic to cancer cells harboring KRAS or BRAF mutations by targeting GAPDH
Authors: Yun Jihye , Kavalier Adam , Mullarky Edouard , Bosch Kaitlyn , Roper Jatin , Rago Carlo , Paik Jihye , Asara John , Gross Steven , Vogelstein Bert , Papadopoulos Nickolas , Cantley Lewis
Published: May 2015

Although more than half of colorectal cancer patients have either KRAS or BRAF mutations, targeted therapies for these subgroups of cancer patients are still lacking. Here, we report that KRAS or BRAF mutant cells exhibit a significant increase in the uptake of oxidized vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased uptake of DHA in the mutant cell lines causes oxidative stress as intracellular DHA is immediately reduced back to vitamin C by reduced glutathione. Elevated reactive oxygen species inactivate GAPDH and thus divert glycolytic flux into the pentose phosphate pathway. Because KRAS or BRAF mutant cells are highly dependent on glycolysis, this altered glucose metabolism induced by vitamin C ultimately leads to an energy deficit in KRAS or BRAF mutant cells and thereby cell death. There are currently a growing number of clinical trials in phases I/II examining the effect of high dose vitamin C as a treatment for cancers, including colorectal cancers. Our results provide a rationale for the pharmacological use of vitamin C as a therapeutic agent to treat colorectal cancer patients with oncogenic KRAS or BRAF mutations.

Citation Format: Jihye Yun, Adam Kavalier, Edouard Mullarky, Kaitlyn Bosch, Jatin Roper, Carlo Rago, Jihye Paik, John Asara, Steven Gross, Bert Vogelstein, Nickolas Papadopoulos, Lewis Cantley. Vitamin C is selectively toxic to cancer cells harboring KRAS or BRAF mutations by targeting GAPDH. [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 2951. doi:10.1158/1538-7445.AM2014-2951
Abstract 2954: A noncanonical mechanism for BCL-2 inhibition of pro-apoptotic BAX
Authors: Barclay Lauren A. , Lee Susan , Stewart Michelle L. , Braun Craig R. , Bird Gregory H. , Walensky Loren D.
Published: May 2015

In response to cellular stress, pro-apoptotic BCL-2 family members such as BAX initiate apoptosis by forming toxic pores in the mitochondrial outer membrane. It is well established that BAX is inhibited by BCL-2 through a sequestering interaction between a surface groove on BCL-2 and the alpha-helical BH3 death domain of BAX. Indeed, BCL-2 overexpression is a cardinal mechanism exploited by cancer cells to resist apoptosis. Novel BCL-2 inhibitors, such as ABT-199, are showing promise as pharmacologic activators of apoptosis in BCL-2-dependent cancers. Emerging data suggest that a distinct interaction between the BH4 domain of BCL-2 and BAX may also account for BCL-2 inhibition of BAX. To dissect this putative interaction mechanism, we generated stabilized alpha-helices of BCL-2 domains (SAHBs) modeled after the BH4 motif of BCL-2 and assessed their capacity to inhibit BAX. We find that distinct BCL-2 BH4 SAHBs directly engage BAX and block BAX-mediated membrane poration. Mutagenesis studies defined key residues for the inhibitory activity of BCL-2 BH4 SAHBs and confirmed the sequence-dependent specificity of action. We extended our findings to full-length BCL-2, demonstrating that point mutagenesis within the BH4 domain disrupts co-immunoprecipitation of expressed BCL-2 and BAX proteins. Taken together, we find that a noncanonical interaction between BCL-2 and BAX contributes to the functional regulation of these critical apoptotic proteins and may serve as a new and complementary target for therapeutic reactivation of cell death in human cancer.

Citation Format: Lauren A. Barclay, Susan Lee, Michelle L. Stewart, Craig R. Braun, Gregory H. Bird, Loren D. Walensky. A noncanonical mechanism for BCL-2 inhibition of pro-apoptotic BAX. [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 2954. doi:10.1158/1538-7445.AM2014-2954
Abstract 2956: The role of apoptotic blebbing in tissue homeostasis and cancer
Authors: Julian Linda , Olson Michael F.
Published: May 2015

Efficient apoptotic cell clearance prevents the release of potentially immunogenic intracellular contents into the surrounding environment to maintain tissue homeostasis; however, the importance of membrane blebbing for apoptotic cell recognition and clearance has not been established. To address fundamental questions regarding the influence of ROCK1 cleavage and consequent apoptotic membrane blebbing in cellular corpse clearance and the maintenance of tissue homeostasis, mice were generated with a single amino acid substitution in the caspase cleavage site (D1113A) that converts ROCK1 to a non-cleavable (ROCK1nc) form. We previously showed that during apoptosis, caspase cleavage of ROCK1 removes an auto-inhibitory carboxy-terminal region to yield a constitutively active kinase fragment that leads to phosphorylation of downstream targets which promote contractile force generation leading to shrinkage, blebbing and nuclear disintegration. When apoptosis was induced in mouse embryonic fibroblasts, the biochemical apoptotic programme itself was unaffected, although cells had significantly impaired morphological alterations. Furthermore, by imaging apoptosis of GFP labelled melanoblasts in ex vivo embryonic skin explants, the effect of the ROCK1nc point mutation was to impair typical apoptotic morphological changes. To understand the biological role of membrane blebbing in the maintenance of tissue homeostasis, we used the liver-selective genotoxic compound diethylnitrosamine (DEN) to induce acute tissue damage. Following DEN treatment, we found that TUNEL positive apoptotic cells were inefficiently cleared in ROCK1nc mice, which resulted in increased neutrophil infiltration in response to the accumulating apoptotic debris. Histologically, the ROCK1nc livers appeared more damaged with evidence of steatohepatitis and perivenular damage, which was accompanied by higher serum alanine transferase levels. We then asked whether defects in tissue damage responses in ROCK1nc mice would affect tumor development in a model of DEN-induced hepatocellular carcinoma. Interestingly, ROCK1nc mice had reduced tumor volume and numbers, suggesting that defective blebbing may be beneficial for reducing tumor initiation and growth. Taken together, our results indicate that efficiency of phagocytic clearance may contribute to cancer susceptibility.

Citation Format: Linda Julian, Michael F. Olson. The role of apoptotic blebbing in tissue homeostasis and 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 2956. doi:10.1158/1538-7445.AM2014-2956
Abstract 2962: Loss of the E3 ubiquitin ligase HACE1 results in enhanced Rac1 signaling contributing to breast cancer progression
Authors: Goka Erik T. , Lippman Marc E.
Published: May 2015

The transition from ductal carcinoma in situ (DCIS) to invasive disease is a crucial step in breast cancer progression. The specific alterations that govern this transition have not been elucidated. HER2/neu overexpression is frequently seen (50-60%) at the DCIS stage but is significantly less common in invasive breast cancer (20-30%) suggesting that additional alterations cooperate with HER2/neu to fully transform mammary epithelial cells. We identified novel genes that are capable of cooperating with HER2/neu allowing full malignant transformation, we used a transgenic mouse model that expresses wild-type neu and performed an insertional mutagenesis screen to identify genes whose alterations induce anchorage-independent growth on primary mouse mammary epithelial cells, an in vitro tumor formation assay. We identified the HECT and ankyrin domain containing E3 ligase 1 (HACE1) as a breast cancer tumor suppressor gene whose loss contributes to the transformation process. Loss of HACE1 expression is commonly seen in breast cancer patients and established breast cancer cell lines supporting the role of HACE1 as a breast cancer tumor suppressor gene. HACE1 ubiquitylates the activated GTP-bound form of the Rho family GTPase Rac1, resulting in ubiquitin mediated degradation of Rac1 via the 26S proteasome. The loss of HACE1 results in the accumulation of activated Rac1 leading to enhanced clonogenicity, migration, and invasion of human mammary epithelial cells (HMECs). In addition, the overexpression of HER2 results in activation of Rac1 levels which is further enhanced upon HACE1 loss in HMECs that further enhance colony formation in soft agar, migration, invasion and allows tumor formation in mice. Importantly, knocking down Rac1 using RNA interference or targeting RAC1 using the small molecule inhibitor EHT 1864 can rescue the effects of HACE1 loss mediated transformation. The results establish HACE1 as a breast cancer tumor suppressor gene by attenuating Rac1 signaling. In addition, our work further supports the role of Rac1 as a critical signaling node in breast cancer and that loss of HACE1 leads to enhanced Rac1 signaling that may contribute to breast cancer progression. These results suggest that HACE1 loss may identify precancerous lesions capable of becoming malignant as well as identify cancers susceptible to RAC targeted therapies.

Citation Format: Erik T. Goka, Marc E. Lippman. Loss of the E3 ubiquitin ligase HACE1 results in enhanced Rac1 signaling contributing to breast cancer progression. [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 2962. doi:10.1158/1538-7445.AM2014-2962
Abstract 2966: Targeting defective DNA repair as a novel chemoprevention strategy for BRCA1-mutated breast cancer
Authors: Alli Elizabeth , Solow-Cordero David , Casey Stephanie C. , Ford James M.
Published: May 2015

Carriers of germline mutations in the Breast Cancer Susceptibility Gene 1 (BRCA1) have an increased risk for developing breast cancer. Unfortunately, BRCA1-mutated cancers are not amenable to current chemoprevention options, often associate with an aggressive clinical course, and thus, are in need of an effective prevention strategy. We previously found that BRCA1 plays a role in DNA base-excision repair (BER) of oxidative DNA damage, and that BRCA1-mutated breast cancers exhibit a compromised ability for BER of oxidative DNA damage. Given that excessive oxidative DNA damage leads to tumorigenesis, we hypothesized that small molecules may be used to enhance the repair of oxidative DNA damage, and in turn, prevent tumorigenesis of BRCA1-mutated breast cancer cells. First, a high-throughput chemical screen identified small molecules that enhance BER of oxidative DNA damage in the presence of mutant BRCA1. These molecules have been termed DNA repair-activating agents. At least two DNA repair-activating agents significantly enhanced BER in mutant BRCA1 but not wild-type BRCA1 cell lines. These molecules also decreased basal levels of oxidative DNA damage as determined by flow cytometry using a FITC-conjugated 8oxoG-binding protein and decreased H2O2-induced oxidative DNA damage as determined by the alkaline comet assay modified for detection of oxidized lesions. Both DNA repair-activating agents directly activated BER, rather than indirectly as a result of induction of DNA damage, as evidenced by the alkaline comet assay for DNA strand breaks. Both agents also showed no cytotoxicity at concentrations that enhanced BER of oxidative DNA damage, which is ideal for chemoprevention. Finally, at least one of the DNA repair-activating agents decreased BRCA1-associated tumorigenesis in vitro and in vivo. The DNA repair-activating agent decreased anchorage-independent growth of BRCA1-mutant/deficient cells without a significant effect on cell viability, as well as delayed tumor formation and decreased tumor burden in a dose-response manner in a xenograft mouse model. Taken together, these data suggest that enhancing DNA base-excision repair of oxidative DNA damage may be a novel strategy for the targeted chemoprevention of BRCA1-associated breast cancers.

Citation Format: Elizabeth Alli, David Solow-Cordero, Stephanie C. Casey, James M. Ford. Targeting defective DNA repair as a novel chemoprevention strategy for BRCA1-mutated breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2966. doi:10.1158/1538-7445.AM2014-2966
Abstract 2974: RNAi mouse models: Revolutionizing drug discovery in vivo
Authors: Premsrirut Prem K. , Yoon BoYoung , Pesic Marina , Dow Lukas E. , Zuber Johannes , Lowe Scott W. , Hannon Gregory J. , Zender Lars , Fellmann Christof
Published: May 2015

RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi tools including RNAi transgenic mice remains a significant limitation. One main hurdle is the identification of potent RNAi triggers, or short hairpin RNAs (shRNAs), that will induce stable and regulated gene silencing. Due to the lack of understanding of the requirements for shRNA biogenesis and target suppression, many predicted shRNAs fail to efficiently induce gene suppression. We have developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale and show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. In addition, we have engineered a new miRNA scaffold, miR-E, that is more efficiently processed and thus produces more potent knockdown of target genes than our previous miR30 system. By combining our sensored miR-E based shRNAs with high efficiency ES cell targeting, we have developed a fast, scalable pipeline for the production of shRNA transgenic mice with reversible gene silencing. We show that RNAi can cause sufficient knockdown to recapitulate the phenotypes of knockout mice, particularly in cancer models. More importantly, unlike traditional knockout models, RNAi has the powerful advantage of reversibility, since the endogenous gene remains intact. Using this system, we generated a number of inducible RNAi transgenic lines and demonstrate how this approach can identify predicted phenotypes and also unknown functions for well-studied genes. In addition, through regulated gene silencing we are able to mimic drug therapy in mice without the actual drug molecule, allowing us to determine the therapeutic value and/or toxic effects associated with systemic gene suppression. Using this approach, we have been able to pinpoint potential toxicities associated with gene inhibition, results that will guide drug development to avoid target failures which will likely cause harmful and intolerable effects in patients. In a model of hepatocellular carcinoma, we demonstrate that short-term inhibition of a ribosomal protein is sufficient to induce stable cell cycle arrest of liver tumor cells. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene - mice with enormous predictive power that will shape our development of better tolerated therapies.

Citation Format: Prem K. Premsrirut, BoYoung Yoon, Marina Pesic, Lukas E. Dow, Johannes Zuber, Scott W. Lowe, Gregory J. Hannon, Lars Zender, Christof Fellmann. RNAi mouse models: Revolutionizing drug discovery in vivo. [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 2974. doi:10.1158/1538-7445.AM2014-2974