bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1031: The effects of NT1014, a novel AMPK activator, on ovarian cancer cell proliferation and apoptosis.
Authors: Kilgore Joshua , Jackson Amanda L. , Qiu Haifeng , Zhou Chunxiao , Batchelor Kenneth , Gehrig Paola , Bae-Jump Victoria
Published: August 2014

Introduction: Anti-diabetic biguanide drugs, such as metformin, have been shown to have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. NT1014 (NovaTarg Therapeutics) is a novel AMPK activator that enters cells specifically through the organic cation transporter, OCT1. Thus, we sought to assess the effect of NT1014 on cell proliferation and apoptosis in human ovarian cancer cell lines.

Methods: Ovarian cancer cell proliferation was assessed by MTT assay after exposure to NT1014. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by Cellometer. Phosphorylated-S6, pan-S6, and OCT1 were evaluated by Western immunoblotting analysis.

Results: NT1014 significantly inhibited proliferation in a dose-dependent manner in both ovarian cancer cell lines (IC50 1000 μM for SKOV3, 500 μM for IGROV1), within 48 hours of exposure. Treatment with NT1014 resulted in G1 cell cycle arrest in both cell lines. Apoptosis occurred in IGROV1 cells exposed to NT1014, but not in SKOV3 cells. Western immunoblot analysis demonstrated that NT1014 decreased phosphorylation of the S6 protein, a key downstream target of the mTOR pathway. Expression of OCT1 was also decreased in a dose-dependent fashion.

Conclusion: NT1014 suppressed ovarian cancer cell growth, predominantly through G1 cell cycle arrest, and resulted in inhibition of the mTOR pathway. More work is needed to determine if metformin and related novel agents, such as NT1014, will be beneficial in the treatment of women with ovarian cancer.

Citation Format: Joshua Kilgore, Amanda L. Jackson, Haifeng Qiu, Chunxiao Zhou, Kenneth Batchelor, Paola Gehrig, Victoria Bae-Jump. The effects of NT1014, a novel AMPK activator, on ovarian cancer cell proliferation and apoptosis. [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 1031. doi:10.1158/1538-7445.AM2013-1031
Abstract 1034: Induction of miR183 and miR21 is associated with down regulation of cellular signaling in nitrogen mustard mediated inhibition of tumor cell proliferation.
Authors: Kim Hong Duck , Shakarjian Michael P. , Vetrano Anna M. , Laskin Jeffrey D. , Heck Diane E.
Published: August 2014

Nitrogen mustard (HN2) is a chemotherapeutic agent used to treat skin cancers and lymphomas including squamous cell carcinomas (SCC) and cutaneous T cell lymphoma. Although HN2 non-selectively alkylates DNA, many tumors exhibit resistance to HN2 therapy. To evaluate additional mechanisms potentially underlying the activity of HN2 we tested the effects of HN2 on Pam 212 murine squamous cell carcinoma cells. We found that mustard treatment reduced cell viability, and that this result was associated with an increase in PKCδ phosphorylation. Furthermore, cytotoxicity was antagonized by the PKCδ selective inhibitor, rottlerin, implicating this PKC isoform in HN2 action. The p38MAPK inhibitor, SB203580, acted in a similar manner to rottlerin, increasing viability 30% in HN2-treated Pam 212 cells. In contrast, PI3kinase inhibitor, wortmannin, enhanced HN2-mediated cytotoxicity, suggesting a protective role for this signaling pathway. Recent reports indicate that the activity of the p38MAPK and PKCδ signaling cascades, pathways involved in regulating glucose uptake and metabolism, growth, motility, and other essential functions for cell survival, is hallmarked by increases in levels of miRNAs 21 and 183. Therefore we next evaluated the effects of HN2 on microRNA (miR) levels using microRNA PCR arrays. In these experiments we found that HN2 treatment enhanced levels of miR21 and miR183 in Pam 212 keratinocytes, effects that were time and dose-dependent dependent. In additional studies we found that transfection of PAM cells with miR21 and miR183 resulted in enhanced sensitivity to inhibitors of PI3 kinase and p38MAPK regulated mTOR activity. Using techniques in real time PCR we observed that HN2-mediated increases in miRNA 21 and 183 levels were inhibited by the mTOR inhibitors 3-MA, rapamycin and LY294002 in PAM cells. Taken together these data indicate that signaling pathways play significant roles in HN2-mediated inhibition of tumor cell proliferation. We speculate that PKCδ, PI3 kinase, p38MAPK and mTOR pathways are important in the anti-cancer activities of HN2 and may be modified in tumors resistant to this agent.

Citation Format: Hong Duck Kim, Michael P. Shakarjian, Anna M. Vetrano, Jeffrey D. Laskin, Diane E. Heck. Induction of miR183 and miR21 is associated with down regulation of cellular signaling in nitrogen mustard mediated inhibition of tumor cell proliferation. [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 1034. doi:10.1158/1538-7445.AM2013-1034
Abstract 1036: Cbl-independent degradation of Met: Ways to avoid agonism of bivalent Met targeting antibody.
Authors: Lee Ji Min , Kim Bogyou , Kim Kyung-Ah
Published: August 2014

The Met receptor tyrosine kinase, found to be constitutively activated in many tumors, has become a leading target for cancer therapy. Disruptions in Met down-regulation have been associated with aggressive tumor progression with several therapeutic strategies addressing this aspect of Met biology. Cbl E3 ligase-mediated degradation, which attenuates Met signaling via ligand-dependent Met internalization, is a major negative regulator of Met expression. It is believed that one of the mechanisms by which the therapeutic anti-Met antibodies induce cancer cell death in Met over-expressing tumors is via internalization and subsequent degradation of Met from the cell surface. However, a previously reported Met targeting antibody demonstrated intrinsic agonistic activity while capable of inducing Cbl-mediated degradation of Met, suggesting that Cbl-mediated degradation requires receptor activation and impedes therapeutic application. We have developed a potent and selective bivalent Met targeting antibody (SAIT301) that invokes Met degradation using an alternative regulator LRIG1. In this report, we demonstrate that LRIG1 mediates degradation of Met by SAIT301 and this degradation does not require Met activation. Furthermore, SAIT301 was able to down-regulate Met and dramatically inhibit growth of tumors with low or no Cbl expression, as well as tumors with Met exon 14 deletion that prevents Met binding to Cbl. In summary, we demonstrate the enhanced therapeutic potential of a novel tumor-inhibiting anti-Met antibody, SAIT301, which utilizes a Cbl-independent, LRIG1-mediated Met degradation pathway and thereby avoids the agonism that limits the effectiveness of previously reported anti-Met antibodies.

Citation Format: Ji Min Lee, Bogyou Kim, Kyung-Ah Kim. Cbl-independent degradation of Met: Ways to avoid agonism of bivalent Met targeting antibody. [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 1036. doi:10.1158/1538-7445.AM2013-1036
Abstract 1041: Sorafenib in combination with rottlerin show an additive effects in renal cell carcinoma (RCC) cells.
Authors: Joeckel Matthias W. , Schneider Elke , Roos Frederik C. , Hampel Christian , Thueroff Joachim W. , Brenner Walburgis
Published: August 2014

Introduction: RCC is a tumor entity with poor outcome. Targeted therapies with tyrosine kinase inhibitors (TKI) are promising agents for combating progression in RCC. We investigated the effect of the TKI sorafenib and sunitinib on cancer progression in vitro. We additionally analysed a coadministration of rottlerin, inhibiting PKCdelta, with sorafenib or sunitinib.

Methods: Four clear cell RCC cell lines were treated with sorafenib or sunitinib alone (5 μM) or in combination with rottlerin (10 μM). Proliferation (BrdU incorporation), apoptosis (caspase 3) and expression of PKCdelta in nuclei (pro-apoptitic), cytoplasm (inactive) and membranous (active) was quantified. The activity of PKCdelta was analysed by detection of phosphorylated PKCdelta (Western blot).

Results: Cell proliferation was reduced by sorafenib and rottlerin, but not by sunitinib. Combination of sorafenib and rottlerin showed an additive effect in proliferation inhibition. Rottlerin and sunitinib induced apoptosis. Althougth sorafenib alone did not induce apoptosis, in combination with rottlerin, apoptosis was more intensely induced than with rottlerin alone. All agents induced an enhanced expression of PKCdelta in the nuclei. Sunitinib in combination with rottlerin reduced membrane -bound PKCdelta. Analogue, expression of activated PKCdelta was reduced after cell treatment with sunitinib in combination with rottlerin.

Conclusion: Coadministration of sorafenib and rottlerin showed an additive effect on proliferation and apoptosis of RCC cells. The expression and activity of PKCdelta was reduced most by sorafenib in comibation with rottlerin. This indicates an improvement in RCC therapy with sorafenib by coadministration of rottlerin.

Citation Format: Matthias W. Joeckel, Elke Schneider, Frederik C. Roos, Christian Hampel, Joachim W. Thueroff, Walburgis Brenner. Sorafenib in combination with rottlerin show an additive effects in renal cell carcinoma (RCC) 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 1041. doi:10.1158/1538-7445.AM2013-1041
Abstract 1043: Connexin might be involved in anti-cancer combination effect of sunitinib and sodium butyrate.
Authors: Uzu Miaki , Sato Hiromi , Suzuki Rina , Okuzawa Hiroko , Siddig Sana , Tuerdi Guligena , Suzuki Sayumi , Nomura Yuki , Sekine Yuko , Yano Tomohiro , Ueno Koichi
Published: August 2014

Sunitinib (SU) is a multi-targeted receptor tyrosine kinase inhibitor (TKI). It is more effective for renal cell carcinoma (RCC) treatment than conventional immunotherapy, but it causes the resistance and severe side effects in some cases. As a combination therapy might be one solution for them, we paid attention to sodium butyrate (NaBu). It is classified into histone deacetylase (HDAC) inhibitor and it is reported HDAC inhibitors can enhance anti-cancer effect of existent drugs including TKI. So, we examined combination effect of SU and NaBu. And to reveal the target of this combination, we examined effects of SU and NaBu on connexin (Cx), which is a component of gap junction (GJ) and tumor suppressors.

We assessed effect of SU and NaBu combination on ACHN (human RCC cell line) cell proliferation by MTT assay and this combination exhibited a synergistic anti-proliferative effect. By real-time PCR, we detected up-regulation of Cx32 mRNA by NaBu. Moreover, NaBu depicted the up-regulated Cx32 protein expression, which was observed by confocal fluorescence microscope. Hemi-channel (Cx hexamer) function was assessed by propidium iodide (PI) uptake into cells. We found both SU and NaBu enhanced PI uptake significantly. GJ function was assessed by scrape-loading. It was confirmed GJ didn't function in ACHN cells and neither SU nor NaBu affected its function. So, it was suggested NaBu could up-regulate Cx32 expression and both SU and NaBu could enhance hemi-channel function. However, we couldn't reveal whether GJ was related to enhancing SU sensitivity. So, we used GJ positive or negative H28 (human malignant mesothelioma cell line) to reveal this matter. As a result, GJ positive cells were more sensitive to SU than negative ones. On the other hand, inhibition of GJ function didn't restore viability of GJ positive cells.

In conclusion, we demonstrated the combination therapy of SU and NaBu had synergistic anti-proliferative effect, so it could be a prominent therapy for RCC. Furthermore, we found Cx could be involved in this combination effect, which might be performed in GJ-independent manner.

Citation Format: Miaki Uzu, Hiromi Sato, Rina Suzuki, Hiroko Okuzawa, Sana Siddig, Guligena Tuerdi, Sayumi Suzuki, Yuki Nomura, Yuko Sekine, Tomohiro Yano, Koichi Ueno. Connexin might be involved in anti-cancer combination effect of sunitinib and sodium butyrate. [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 1043. doi:10.1158/1538-7445.AM2013-1043
Abstract 1046: AS1411 activity is regulated by epidermal growth factor receptor signaling pathway.
Authors: Reyes-Reyes Elsa M. , Salipur Francesca R. , Bates Paula J.
Published: August 2014

The anticancer AS1411 agent is a G-rich phosphodiester oligodeoxynucleotide, which forms a stable quadruplex structure and binds specifically to nucleolin as an aptamer. It efficiently inhibits proliferation and induces cell death in many types of cancer cells, but has little effect on normal cells. We have also shown that AS1411 is taken up by macropinocytosis and stimulates further macropinocytosis by a nucleolin-dependent mechanism in several cancer cells. AS1411 activity correlates with stimulated macropinocytosis, suggesting this hyperstimulation of macropinocytosis may explain the unusual cancer cell death caused by AS1411. Macropinocytosis is a ligand-independent endocytic pathway that is normally activated by growth factor receptor stimulation. On the other hand, nucleolin has been reported that can enhance growth factor signaling by binding directly to some growth factor receptors. Therefore in this study, we determine the participation of growth factor signaling pathway in AS1411-induced macropinocytosis and cell death. Pre-incubation of DU145 cells with a specific EGFR inhibitor, but not PDGFR or JAK2 inhibitors, significantly inhibited the stimulation of AS1411-mediated macropinocytosis, suggesting that AS1411 activates EGFR signaling pathways. This was confirmed by observing that DU145 cells treated with AS1411 induced tyrosine phosphorylation of EGFR and activation of its downstream effectors, including p38, PI3K and Rac1. Furthermore, AS1411 seem to induce the interaction between EGFR and nucleolin in accord with EGFR activation. We also found that the activation of EGFR signaling pathway by AS1411 might regulate its cancer cellular response. Simulation of macropinocytosis by AS1411 was significantly inhibited in DU145 cells pre-treated with pharmacological inhibitors of Rac1 or PI3K, but not by Src inhibitor. Moreover the ability of AS1411 to inhibit cell proliferation and induce cell death was reduced by an EGFR-neutralizing antibody (cetuximab) or by downregulating the expression of Rac1 using siRNAs. These results indicate that AS1411 mediates the activation of EGFR signaling pathway to induce cancer cell death, and suggest that AS1411 might redirect EGFR signaling pathway from a cell survival signal to a cell death signal.

Citation Format: Elsa M. Reyes-Reyes, Francesca R. Salipur, Paula J. Bates. AS1411 activity is regulated by epidermal growth factor receptor signaling pathway. [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 1046. doi:10.1158/1538-7445.AM2013-1046
Abstract 1048: Targeted BRAF therapy attenuates the promotional activity of estrogen in thyroid cancer.
Authors: Hanly Elyse K. , Rajoria Shilpi , George Andrea L. , Suriano Robert , Geliebter Jan , Pantelides Harry , Shin Edward J. , Tiwari Raj K.
Published: August 2014

Thyroid cancer, which is three to four times more prevalent in women than men, is the most common endocrine-related cancer in the United States and its incidence has increased in the past three decades. Among various genetic lesions identified in thyroid cancer, the BRAF V600E mutation is found in 50% of papillary thyroid cancers and affords an opportunity for targeted drug therapy. Our laboratory identified estrogen as a promotional factor in thyroid cancer, as estrogen enhances proliferation, migration, and invasion of thyroid cancer cells in vitro. Since the mitotic activity of estrogen is partially mediated by the mitogen-activated protein kinase (MAPK) pathway, we hypothesized that a targeted BRAF V600E inhibitor, PLX4032, would inhibit estrogen-mediated tumor-promoting activity. Results demonstrate that PLX4032 abrogates estrogen-induced increases in proliferation and invasion of BRAF V600E-mutated BCPAP papillary thyroid cancer cells in a dose-dependent manner. In contrast, this phenomenon was not observed in Nthy-ori 3-1 thyroid cells, which are wild-type immortalized thyroid cells without the BRAF V600E mutation. Expression of estrogen receptor is not modulated by PLX4032 treatment, indicating that PLX4032 targets signal transduction pathways in estrogen-responsive thyroid cells. PLX4032 down-regulates the expression of key signaling molecules of the BRAF pathway, including mitogen-activated protein kinase kinase (p-MEK), extracellular-signal regulated kinases (p-ERK), and mammalian target of rapamycin (p-mTOR) in BRAF-mutated BCPAP cells and does not alter the expression of these signaling molecules in wild-type Nthy-ori 3-1 cells. These results demonstrate a direct interlinking of a genetic lesion amenable to drug therapy with the promotional activity of estrogen. These findings will lead to an evaluation of combinational therapy including anti-estrogens and PLX4032 in preclinical animal models and a subset of patients harboring the BRAF V600E mutation. Targeting growth-promoting signal transduction pathways constitutively activated by genetic lesions will ultimately aid in overcoming resistance to traditional radioactive iodine therapy in patients with specific mutations such as BRAF V600E.

Citation Format: Elyse K. Hanly, Shilpi Rajoria, Andrea L. George, Robert Suriano, Jan Geliebter, Harry Pantelides, Edward J. Shin, Raj K. Tiwari. Targeted BRAF therapy attenuates the promotional activity of estrogen in thyroid 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 1048. doi:10.1158/1538-7445.AM2013-1048
Abstract 1051: EGFR inhibitors attenuate caspase-independent cell death and confer negative effects on cisplatin .
Authors: Yamaguchi Hirohito , Hsu Jennifer L. , Chen Chun-Te , Wang Ying-Nai , Hsu Ming-Chuan , Chang Shih-Shin , Du Yi , Ko How-Wen , Herbst Roy S. , Hung Mien-Chie
Published: August 2014

EGFR tyrosine kinase inhibitors (TKIs) have demonstrated exciting results and led to advanced therapy trials in combination with chemotherapy. However, clinical studies have raised an important question why the antagonism between TKI and chemotherapy occurs in lung cancer patients only with wildtype but not mutant EGFR. In this study, we further explored the mechanism through which gefitinib gives rise to the negative effects when combined with cisplatin in wildtype EGFR lung cancer cells. We found that gefitinib inhibited cisplatin-induced caspase-independent cell death (CID) but not caspase-dependent cell death (apoptosis). In wildtype EGFR cells, gefitinib not only inhibited CID but also failed to induce apoptosis and therefore compromising the efficacy of cisplatin. Moreover, inhibition of the EGFR/ERK/AKT pathway by gefinitib activated FOXO3a and subsequently reduced reactive oxygen species (ROS) induced by cisplatin treatment and ROS-mediated CID. To overcome this, we showed that SAHA and erastin, which are known to induce ROS-mediated CID, strongly enhanced the effect of cisplatin in wildtype EGFR cells. Taken together, our findings provide a possible explanation for the previously failed clinical trials as well as a novel insight into future cancer therapy.

Citation Format: Hirohito Yamaguchi, Jennifer L. Hsu, Chun-Te Chen, Ying-Nai Wang, Ming-Chuan Hsu, Shih-Shin Chang, Yi Du, How-Wen Ko, Roy S. Herbst, Mien-Chie Hung. EGFR inhibitors attenuate caspase-independent cell death and confer negative effects on cisplatin . [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 1051. doi:10.1158/1538-7445.AM2013-1051
Abstract 1052: Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer.
Authors: Hung Huynh T. , Ong Richard , Soo Khee Chee , Puehler Florian , Scholz Arne , Mumberg Dominik , Zopf Dieter , Ziegelbauer Karl
Published: August 2014

Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the Ras/Raf/MEK/ERK signaling cascade plays a critical role in signal transduction pathways that regulate growth factor response, angiogenesis, tumor cell proliferation, and apoptosis, blocking this signaling pathway could thus have therapeutic efficacy. This study aims to target this pathway in preclinical models of GC with refametinib (BAY 86-9766, RDEA119), an orally available, allosteric inhibitor of MEK1/2.

Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with refametinib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or refametinib-treated xenografts were analyzed by immunohistochemistry for cell proliferation, apoptosis and microvessel area, using antibodies against p-Histone H3Ser10, cleaved PARP and CD31, respectively. Tumor lysates were analyzed by Western Blotting with antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis.Results: Refametinib demonstrated significant tumor growth inhibition ranging from 66-87% in eight GC models at a dose of 15 mg/kg/d. Refametinib inhibited cell proliferation by 2-4 fold as observed by reduced staining for p-Histone H3Ser10. Increased staining for caspase-cleaved p85 PARP by 1.5 to 9 fold was detected, indicating that refametinib induced apoptosis. Furthermore the compound significantly inhibited angiogenesis in 2 of 8 models by 3-fold compared to the vehicle-treated group as indicated by reduced CD31 staining. Reduced cell proliferation and induction of apoptosis were associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-p90RSKSer359/363, p-p70S6KThr421/Ser424, p-4EBP1Thr70, p-S6RSer235/236 and p-eIF4ESer209 and elevation of p-AktSer473, Bim and cleaved PARP. Conclusion: We show that single-agent refametinib has significant anti-tumor activity in patient-derived gastric cancer preclinical models. Our findings support further investigation of refametinib either in monotherapy or in combination with inhibitors of the PI3K-AKT-mTOR pathway in the treatment of GC.

Citation Format: Huynh T. Hung, Richard Ong, Khee Chee Soo, Florian Puehler, Arne Scholz, Dominik Mumberg, Dieter Zopf, Karl Ziegelbauer. Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of 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 1052. doi:10.1158/1538-7445.AM2013-1052
Abstract 1055: Regorafenib-mediated potent and reversible inhibition of human hepatocellular carcinoma (HCC) cell growth, migration and AFP.
Authors: Carr Brian I. , D'Alessandro Rosalba , Refolo Maria Grazie , Iacovazzi Minna , Lippolis Catia , Messa Caterina , Cavallini Aldo
Published: August 2014

Background: Most HCC presents at an advanced stage, for which there are few effective treatments. Sorafenib enhanced survival by 10 weeks in a randomized trial and was approved by FDA. Sorafenib is a base for building better treatments. Regorafenib (Fluoro-Sorafenib, BAY 73-4506 or Stivarga) is a more potent analog in current clinical trials.

Methods: HCC cell lines were grown in culture. Growth was assessed using MTT, migration by scratch assay, medium AFP by clinical chemoluminescent immunometric method, cell AFP and kinases by western blot and cell AFP mRNA by real-time PCR.

Results: HCC growth in different cell lines was inhibited by Regorafenib 1-5 μM IC50. Removal of drug from the medium resulted in resumption of normal cell growth even after subsequent sub-culture. This recovery could be partially blocked by low concentrations of vitamin K1 or doxorubicin that were not growth inhibitory when used alone. Regorafenib 1 μM pre-treatment for 2 weeks resulted in very slow recovery. Cell treatment in culture with Regorafenib resulted in early decreases in phospho-ERK levels, followed by return to baseline levels within hours, despite continuous drug exposure. Cell growth recovery after Regorafenib was associated with induction of high phospho-JNK levels and the recovery was blocked by the actions of a JNK inhibitor. Regorafenib also inhibited cell migration and both AFP mRNA by PCR and cellular AFP on WB, and cell culture medium levels of AFP, but at much lower concentrations than needed (0.1-1 μM) for growth inhibition. Removal of drug also resulted in recovery of cell migration, but this recovery could be partially blocked by addition of vitamin K1 or doxorubicin, at drug concentrations that were not themselves inhibitory of cell migration. Even at 1 μM, Regorafenib slowed cell growth and migration after prolonged cell exposure and sub-culture. Pre-treated and recovering cells were much more sensitive to inhibition of migration by low concentration vitamin K1 6.25 μM or doxorubicin 0.01 μM. Conclusions: Regorafenib is a potent inhibitor of HCC cell line growth and works even at very low drug concentrations when cells are exposed for prolonged times. The effects are reversible and JNK could be a critical player in this process. Very low concentrations inhibit HCC cell migration and AFP. Regorafenib appears to be a promising new agent.

Citation Format: Brian I. Carr, Rosalba D'Alessandro, Maria Grazie Refolo, Minna Iacovazzi, Catia Lippolis, Caterina Messa, Aldo Cavallini. Regorafenib-mediated potent and reversible inhibition of human hepatocellular carcinoma (HCC) cell growth, migration and AFP. [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 1055. doi:10.1158/1538-7445.AM2013-1055
Abstract 1062: Imaging DNA damage response (DDR) during oncogenesis.
Authors: Cornelissen Bart , Able Sarah , Kersemans Veerle , Vallis Katherine A.
Published: August 2014

Introduction: DNA damage repair (DDR) signalling is upregulated during tumorigenesis and acts as a p53-mediated block on proliferation. Expression of the DNA double strand break (DSB) repair protein, γH2AX, is induced by the activated kinases pATM or pATR during oncogenesis. Previously, we showed that γH2AX presents an abundant epitope for non-invasive in vivo imaging of DSBs. We used 111In-labelled anti-γH2AX antibodies, conjugated to the cell penetrating peptide, TAT, to non-invasively image DDR during oncogenesis in a genetically engineered mouse model of HER2/neu-overexpression driven breast cancer.

Methods: Balb/neuT mice, genetically modified to express mutated rat neuT under MMTV promotor control, form multiple palpable carcinomas in mammary fat pads when animals are 130 days old. SPECT images were acquired weekly from balb/neuT mice (age 40-140 days; n=23) 24 h after i.v. injection of 111In-labelled anti-γH2AX-Tat or the non-selective agent, 111In-rIgG-Tat using a small animal SPECT/CT scanner. 111In uptake in fat pads was determined using volume of interest analysis. 111In uptake of >5%ID/g was deemed positive. Anatomical and histological changes were assessed at different ages using DCE-MRI imaging and immunohistochemistry.

Results: Balb/neuT mice that were 80 days old developed multiple hyperplastic and dysplastic lesions in mammary fat pads. γH2AX foci were observed in these regions in balb/neuT mice that were 80-100 days old (10-15 foci/cell), but were less numerous in overt tumor tissue in 130-day-old mice (4-5 foci/cell). γH2AX foci were not observed in normal tissues in older mice (0 foci/cell), in mammary fat pads in younger mice (0 foci/cell), or in wild type mice (0 foci/cell). Serial SPECT imaging revealed a 7-fold increase in 111In-anti-γH2AX-Tat accumulation in mammary fat pads in mice 80-110 days old (up to 8.5 %ID/g), compared to non-specific control (P=0.0007), but not in younger or older mice (P>0.05). Repeated SPECT imaging itself did not influence tumor occurrence (P>0.05). The median time to detection of positive tissue was 96 days, much sooner than detection of lesions >150 μm by DCE-MRI (117 days), or palpation (130 days) (P<0.0001).

Conclusion: Using a γH2AX-targeted radiopharmaceutical, we were able to image the DNA damage response during neuT-mediated oncogenesis in balb/neuT mice. DDR imaging identified neoplastic tissue significantly earlier than anatomical imaging. These results suggest that γH2AX imaging has potential as a screening tool for cancer-prone individuals.

Citation Format: Bart Cornelissen, Sarah Able, Veerle Kersemans, Katherine A. Vallis. Imaging DNA damage response (DDR) during oncogenesis. [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 1062. doi:10.1158/1538-7445.AM2013-1062
Abstract 1069: Vav3 enhances androgen receptor splice variant activity and is critical for castration resistant prostate cancer growth and survival.
Authors: Peacock Stephanie , Fahrenholtz Cale D. , Burnstein Kerry L.
Published: August 2014

Advanced or metastatic prostate cancer is treated by androgen deprivation; however, patients inevitably relapse with castration resistant prostate cancer (CRPC) that grows independently of androgen. However, CRPC remains dependent on androgen receptor (AR) signaling, which includes constitutive, ligand–independent action of naturally occurring AR splice variants. For example, the AR splice variant AR3 (also termed AR–V7) is expressed in CRPC and is linked to poor prognosis. Vav3, a Rho GTPase guanine nucleotide exchange factor, is an AR coactivator that is up–regulated in human prostate cancer as compared to benign tissue as well as in pre–clinical models of CRPC. Vav3 confers castration–resistant growth to androgen–dependent human prostate cancer cells and increased expression has been linked to increased clinical biochemical relapse. Despite the importance of AR coactivators in promoting CRPC, the potential role of these regulatory proteins in modulating AR splice variant activity is unknown. We examined the contributions of Vav3 to AR activity in two CRPC cell lines that naturally express relatively high levels of Vav3 and AR3. Vav3 or AR3 knockdown greatly attenuated cell proliferation, soft agar growth and ligand–independent AR activity. Vav3 potently enhanced the transcriptional activity of AR3 and another clinically relevant AR splice variant ARv567es. Furthermore, Vav3 enhanced AR splice variant activity through a distinctly different mechanism than Vav3 coactivation of full length AR. Vav3 knockdown resulted in lowered nuclear AR3 levels while total AR3 protein and mRNA levels remained static. Conversely, overexpression of Vav3 resulted in increased nuclear AR3. Co–immunoprecipitation revealed that AR3 and Vav3 interact. These novel data demonstrating physical and functional interactions between Vav3, a unique AR coactivator, and an AR splice variant provide insights into the mechanisms by which Vav3 exploits and enhances AR signaling in the progression to CRPC.

Citation Format: Stephanie Peacock, Cale D. Fahrenholtz, Kerry L. Burnstein. Vav3 enhances androgen receptor splice variant activity and is critical for castration resistant prostate cancer growth and survival. [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 1069. doi:10.1158/1538-7445.AM2013-1069
Abstract 107: Pre-diagnostic levels of serum one-carbon metabolism biomarkers and risk of hepatocellular carcinoma.
Authors: Butler Lesley M. , Wang Renwei , Bottiglieri Teodoro , Gao Yu-Tang , Yuan Jian-Min
Published: August 2014

Pre-diagnostic levels of serum one-carbon metabolism biomarkers and risk of hepatocellular carcinoma

BACKGROUND. It is well established that rats fed diets deficient in choline and methionine spontaneously develop hepatocellular carcinoma (HCC). Tumor DNA from these animals show a characteristic hypomethylation pattern, suggesting that disruption of the one-carbon metabolism (1CM) pathway may be an underlying mechanism for hepatocarcinogenesis. Prospective epidemiological data on circulating levels of 1CM biomarkers in relation to HCC risk have been lacking.

METHODS. We prospectively examined the association between prediagnostic serum concentrations of betain, choline, cystathionine, homocysteine (Hcy), methionine, 5-methyltetrahydrofolate (5-MTHF), pyridoxal-5-phosphate (PLP, the bioactive form of vitamin B6), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and risk of developing HCC among 297 incident HCC cases and 631 matched controls from a cohort of 18,244 men in Shanghai, China. Conditional logistic regression method was used to calculate odds ratios (OR) and 95% confidence intervals (CI) adjusted for smoking, alcohol use, hepatitis B surface antigen (HBsAg) seropositivity and chronic liver disease.

RESULTS. Higher serum levels of PLP were associated with statistically significantly reduced risk of HCC. The ORs (95% CIs) for the 2nd, 3rd, and 4th quartiles of serum PLP were 0.52 (0.32, 0.85), 0.57 (0.35, 0.93) and 0.35 (0.20, 0.60), respectively, compared with the 1st quartile (Ptrend<0.01). Among men with positive HBsAg, doubling serum choline concentration was associated with a statistically significant 58% decrease in HCC risk (OR=0.42; 95% CI: 0.19, 0.92) and doubling serum methionine concentration was positively associated with HCC risk (OR=2.94; 95% CI: 1.27, 6.82). Serum levels of cystathionine were associated with increased risk of HCC among all subjects (OR=1.92; 95% CI: 1.14, 3.21; Ptrend=0.01, comparing the 4th to 1st quartile). There was no association for HCC risk with circulating levels of 5-MTHF, Hcy, SAM, or SAH.

CONCLUSION. Choline and PLP may be protective against the development of HCC whereas circulating methionine may be a risk factor for developing HCC, especially among high-risk individuals. Future investigation is warranted to test whether diets high in vitamin B6 are effective in preventing HCC development in high-risk populations.

Citation Format: Lesley M. Butler, Renwei Wang, Teodoro Bottiglieri, Yu-Tang Gao, Jian-Min Yuan. Pre-diagnostic levels of serum one-carbon metabolism biomarkers and risk of hepatocellular 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 107. doi:10.1158/1538-7445.AM2013-107
Abstract 1071: Impact of AR activation status on the DNA damage response.
Authors: Goodwin Jonathan F. , Schiewer Matthew J. , Feng Felix Y. , Knudsen Karen E.
Published: August 2014

Recent evidence implicates the androgen receptor (AR) as a critical modulator of the DNA damage response in prostate cancer. Treatment for locally advanced disease depends on combined radiation therapy and ablation of AR function, and clinical evidence strongly supports the contention that active AR reduces the response to radiotherapy. Moreover, it was reported in vitro that AR activation can induce DNA damage at sites of active transcription, thus providing additional evidence for crosstalk between the DNA damage response and AR pathways. Despite these observations, the underpinning mechanism by which AR alters the response to DNA damage remains unknown.

Several lines of evidence will be discussed which illuminate a role for AR in the response to DNA damage. First, investigation in a battery of hormone-therapy sensitive prostate cancer cells (in which AR activity remains dependent on ligand binding) and in castrate resistant tumor cells (reflecting advanced disease wherein AR activity is enhanced) showed that androgen depletion enhances the response to ionizing radiation. Restoration of dihydrotestosterone reduced the response to radiation, thus demonstrating that ligand-induced AR activity promotes a resistance to radiation. Second, the mechanism by which AR promotes radioresistance was addressed. Preliminary findings indicate that in the presence of DNA damage, AR dramatically alters the rate of DNA damage repair independent of significant effects on cell cycle. Moreover, these studies uncovered an unexpected role for androgen-activated AR to alter expression and subsequent activity of genes critical for the DNA damage response, possibly through differential recruitment of AR to loci of key players in the response to damage. Finally, a series radioresistant prostate cancer model systems were developed, wherein the impact of AR on acquired radioresistance is under investigation; early studies in these models indicate that altered AR output is associated with acquired radioresistance. Combined, the studies to be presented reveal novel functions of androgen and AR in controlling the molecular and cellular response to DNA damage, and provide the basis for future studies directed at targeting DNA-damage specific AR activity in the course of human disease.

Citation Format: Jonathan F. Goodwin, Matthew J. Schiewer, Felix Y. Feng, Karen E. Knudsen. Impact of AR activation status on the DNA damage response. [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 1071. doi:10.1158/1538-7445.AM2013-1071
Abstract 1075: Steroid receptor reprogramming of the chromatin landscape: crosstalk at the genomic level.
Authors: Miranda Tina B. , Voss Ty C. , Sung Myong-Hee , Baek Songjoon , John Sam , Hawkins Mary , Grontved Lars , Schiltz R. Louis , Hager Gordon L.
Published: August 2014

Crosstalk between the estrogen receptor (ER) and the glucocorticoid receptor (GR) plays an important role in controlling many cellular processes. Physiological interactions between ER and GR are not only important for the development of certain tissues, such as the uterus and bone, but also may play an important role in breast cancer. Recent studies have shown that the GR and ER status in breast cancer is a significant factor for determining the outcome of the disease. However, the mechanistic details defining the cellular interactions between ER and GR are poorly understood. Since the regulation of receptor binding to response elements controls the transcriptional output in response to hormones, it is logical to suspect that co-treatment of cells with corticosteroids and estradiol would have an effect on the genome-wide binding landscapes for GR and ER. Therefore, using chromatin immunoprecipitation followed by high-throughput sequencing we investigated the changes in genome-wide binding of ER and GR upon co-activation and revealed that there is global re-arrangement of steroid receptor binding. Furthermore, we unveiled a novel mechanism dictating the molecular interplay between ER and GR. Genome-wide sequencing of DnaseI hypersensitive sites upon induction of GR shows that GR facilitates selective access of ER to specific sites in the genome by maintaining an accessible configuration at these response elements. In addition, activation of ER can affect chromatin structure at novel GR binding sites, resulting in a new class of GR binding elements. Further studies show that at the sites where ER binding is facilitated by GR, binding to these newly accessible sites is not dependent on the direct binding of ER to its response element. Instead ER is brought to these sites through interactions with other factors, such as AP1. Overall, these studies define genome level interactions between ER and GR whereby the activation of multiple steroid receptors has a dramatic impact on controlling which regulatory elements are accessible to each receptor. The unveiling of this mechanism is important for understanding the molecular interplay between ER and GR in development and in cancer and may represent a general mechanism for crosstalk between receptors.

Citation Format: Tina B. Miranda, Ty C. Voss, Myong-Hee Sung, Songjoon Baek, Sam John, Mary Hawkins, Lars Grontved, R. Louis Schiltz, Gordon L. Hager. Steroid receptor reprogramming of the chromatin landscape: crosstalk at the genomic level. [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 1075. doi:10.1158/1538-7445.AM2013-1075
Abstract 1077: Dominant negative PI3-Kinase inhibits UV-induced squamous cell carcinoma in vivo.
Authors: Dickinson Sally E. , Olson Erik R. , Rusche Jadrian J. , Criswell P. Jane , Zhang Jack , Dong Zagong , Hu Chengcheng , Saboda Kathylynn , Alberts David S. , Bowden G. Timothy
Published: August 2014

Phosphoinositide-3 Kinase (PI3-Kinase) is an upstream regulator of cellular proliferation and anti-apoptotic signaling that has been implicated in cancer induction in many tissues, including skin. Pharmacological inhibition of PI3-Kinase is effective at inhibiting both chemical and UV-driven skin carcinogenesis. However, the specificity of these pharmacological inhibitors is often questioned. Therefore, specific genetic inhibition of PI3-Kinase in the epidermis needs to be carried out in mice. We report here on the generation of a new transgenic mouse which constitutively expresses the dominant negative mutant form of the regulatory subunit of the PI3-Kinase dimer, p85, in the epidermis under control of the keratin-5 promoter. These p85DN transgenic mice were derived on the SKH-1 hairless genetic background. A chronic UVB exposure experiment showed significant inhibition of both tumor size (p=0.0012) and tumor burden (p=0.0003) for mice carrying the transgene compared to wildtype littermates. The time to first tumor was also delayed in transgenic mice, but with only marginal significance (p=0.07). p85DN mice also showed significant inhibition of epidermal Activator Protein-1 (AP-1) transcription factor activity after acute UVB treatment (p=0.046). UVB induced activation of AP-1 in the epidermis has been shown to play a functional role in UVB mouse skin carcinogenesis. Further analysis of protein expression after acute treatment with UVB suggests that Akt signaling and COX-2 activation are both downregulated in p85DN transgenic mice. To our knowledge, this is the first report of genetic inhibition of PI3-Kinase in mouse skin carcinogenesis studies. Together, these data suggest a functional role of PI3-Kinase in UVB-induced squamous cell carcinoma and support the assertion that inhibition of this pathway is beneficial in chemoprevention of non-melanoma skin cancer. This work was supported in part by the following NIH grants: K07CA132956, K07CA132956-02S1, P01CA27502 and P30CA23074.

Citation Format: Sally E. Dickinson, Erik R. Olson, Jadrian J. Rusche, P. Jane Criswell, Jack Zhang, Zagong Dong, Chengcheng Hu, Kathylynn Saboda, David S. Alberts, G. Timothy Bowden. Dominant negative PI3-Kinase inhibits UV-induced squamous cell carcinoma in vivo. [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 1077. doi:10.1158/1538-7445.AM2013-1077
Abstract 1085: MYC inhibition is a potent therapy against glioma and induces mitotic crisis in cancer cells.
Authors: Annibali Daniela , Whitfield Jonathan R. , Favuzzi Emilia , Jauset Toni , serrano Erika , Folch Gerard , Cuartas Marta I. , Gonzalez Alba , Brown Swigart Lamorna , Nasi Sergio , Evan Gerard I. , Seoane Joan , Soucek Laura
Published: August 2014

Gliomas are the most common primary tumors affecting the adult human Central Nervous System (CNS). The most lethal is grade IV glioblastoma (GBM), which gives a median survival of only 15 months, but less severe grades also respond poorly to standard therapy. Myc is a bHLHZip transcription factor, causally implicated in most human tumors. In the past, we employed a dominant negative of Myc transactivation activity, termed Omomyc, and we showed that Myc inhibition is a potent strategy in cancer therapy, both in K-RasG12D-driven lung tumors and in T antigen-driven pancreatic β-cell insulinomas. Now, we make use of a mouse model of Ha-Ras driven glioma coupled with our Omomyc switchable model in order to assess Myc inhibition as a therapeutic strategy in glioma. Myc inhibition has a dramatic therapeutic impact in the animals, being able to both prevent formation and cause regression of tumors. We also tested neuroprogenitor cells derived from the same animal model as putative cells of origin of glioma. In this context, Myc inhibition reduced proliferation, increased death and impaired the self-renewal of Ha-Ras transformed neuroprogenitors. Similar results were obtained with human glioblastoma cell lines, which presented severe mitotic catastrophe as a consequence of Omomyc expression, revealing a new Achilles’ heel of glioblastoma. Finally, Myc inhibition was tested in orthotopic xenografts using patient-derived tumor samples and showed once again dramatic therapeutic impact, conferring a significant survival advantage to recipient animals. Conclusion: Myc inhibition is a potent therapeutic strategy in glioma and its effects include induction of mitotic crisis in tumor cells.

Citation Format: Daniela Annibali, Jonathan R. Whitfield, Emilia Favuzzi, Toni Jauset, Erika serrano, Gerard Folch, Marta I. Cuartas, Alba Gonzalez, Lamorna Brown Swigart, Sergio Nasi, Gerard I. Evan, Joan Seoane, Laura Soucek. MYC inhibition is a potent therapy against glioma and induces mitotic crisis in 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 1085. doi:10.1158/1538-7445.AM2013-1085