bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2447: Novel TLR4-Nanog stemness pathway in liver cancer stem cells confers resistance to TGF-β-mediated tumor suppression through YAP1 and IGF2BP3-AKT-mTOR
Authors: Machida Keigo , Chen Chia-Lin , Kashiwabara Claudine , Liu Jian-Chang , Feldman Douglas , Hyeongnam Jeong Joseph , Tsukamoto Hidekazu
Published: June 2012

Cancer stem cells (CSCs) confer resistance to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC) commonly observed in hepatitis C virus (HCV)-infected patients with alcohol abuse. We recently identified pluripotency marker Nanog is induced by Toll-like receptor 4 (TLR4) signal activated by alcohol-associated endotoxemia in CD133+/CD49f+ CSCs from liver cancers in HCV Ns5a transgenic mice fed alcohol. We propose the novel oncogenic TLR4 signaling reciprocally regulates tumor suppressive TGF-β pathway. Molecular mechanisms for the genesis of CSCs through TLR4 signaling and defective TGF-β pathway, however, are unknown. CD133+/CD49f+ CSCs were isolated from liver tumors of alcohol-fed HCV Ns5a Tg mice. Lentiviral cDNA library were established from the CSCs for screening genes responsible for defective TGF-β signaling. GFP-labeled CSCs were injected in nude or NOG mice with or without genetic manipulation of TGF-β signaling components or newly identified genes to monitor tumor growth. Interactions of proteins encoded by identified genes with TGF-β pathway components were examined. Here we demonstrate TLR4/Nanog-dependent CSCs are indeed defective in TGF-β signaling. Functional oncogene screening of a cDNA library identified the organ size control pathway target Yap1 and AKT activator Igf2bp3 as Nanog-dependent genes that inhibit TGF-β signaling in CSCs through Yap1/Smad7/Smad3 association and suppressed Smad3 phospho-activation due to AKT-mTOR pathway, respectively. Silencing of both Yap1 and Igf2bp3 restores TGF-β signaling, inhibits pluripotency gene expression, and sensitizes CSCs to autophagy and cell death by the conventional chemotherapy (rapamycin and sorafenib). Therefore, a combined therapy targeting both YAP1 and AKT-mTOR pathways to restore TGF-β pathway should improve therapeutic efficacy for HCC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2447. doi:10.1158/1538-7445.AM2011-2447
Abstract 2463: Side population cells have some stem-like properties in hepatocellular carcinoma
Authors: okabe Hirohisa , Beppu Toru , Ishiko Takatoshi , Chikamoto Akira , Masuda Toshiro , Otao Ryu , Hasita Horlad , Mima Kosuke , Takamori Hiroshi , Baba Hideo
Published: June 2012

Background: There are many problems to be solved in cancer stem cells (CSCs) in digestive organs. Liver cancer stem cells also have a major problem that we have to determine the cancer stem cell markers and clarify the function of CSCs. In this study, we tried to validate CSC markers that are previously reported using side population (SP) cells in hepatocellular carcinoma cell lines.

Methods: Side population cells in seven hepatocellular carcinoma (HCC) cell lines (PLC/PRF/5, HuH1, HuH7, HLE, HepG2, HLF, and SK-Hep1) were analyzed using Flow cytometry (FACS AriaII). The differences between SP cells and Non-SP cells in several markers relating to epithelial-mesenchymal transition and in several liver CSC markers were investigated using quantitative RT-PCR. The difference about intracellular signals between them was also studied using Western blot analysis. Using HuH7 cell, the ability of spheroid formation of SP cells and the difference between spheroid forming cells and 2-dimensional cultured cells were investigated.

Results: The range of percentage of SP cells was from 0 % to 4.1 %. We sorted SP cells from four HCC cell lines (PLC/PRF/5, HuH1, HuH7, and HLE). There were no remarkable difference of CDH1 and vimentin between SP cells and Non-SP cells. Previously reported liver cancer stem cell markers such as EpCAM, CD90, and CD44 were significantly increased in SP cells. However, the expression of CD133 and Bmi1 in SP cells was unexpectedly decreased or similar to non-SP cells. The ability of spheroid formation of SP cells was significantly higher than that of non-SP cells. Spheroid forming cells showed remarkably higher expression of EpCAM, CD90, and CD44 than 2-dimensional cultured cells. These were considered to be important markers regulating CSCs. The expression of cell cycle marker MIB1 in the SP cells was remarkably decreased compared to that of non-SP cells. This fact supports to the theory that normal or cancer stem cells maintain their cell cycle arrest. SP cells maintained specific signals activated such as sonic hedgehog and MAPK signals.

Conclusions: Liver cancer stem cell markers such as EpCAM, CD90, and CD44 were considered to be important for the maintenance of SP cells in hepatocellular carcinoma. The activation of specific intracellular signals was also seen in SP cells. We are now challenging to confirm the importance of those signals using HCC cells extracted from human liver tissues. The current study may be help of understanding the regulation of liver cancer stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2463. doi:10.1158/1538-7445.AM2011-2463
Abstract 2482: Bat3 facilitates histone H3 lysine 79 dimethylation by DOT1L to promote ionizing radiation-induced 53BP1 foci formation during G1- and G2-phases of the cell cycle
Authors: Wakeman Timothy P. , Feng Junjie , Wang Xiao-fan
Published: June 2012

DOT1L is a unique histone methyltransferase involved in normal cellular metabolism and in the initiation of certain leukemias. DOT1L specifically methylates histone H3 at K79, promoting the interaction of H3 with various binding partners. 53BP1, a component of the DNA damage response machinery, has been shown to interact with H3 K79-diMe following exposure to ionizing radiation; however, it remains unclear if this modification is required for 53BP1 foci formation as 53BP1 interacts with histone H4 K20-diMe with a much higher efficiency. Here, we clarify the role of H3 K79-diMe by demonstrating that this modification contributes to IR-induced 53BP1 foci formation during G1- and G2-phases of the cell cycle, but is negligible during S-phase. Further, we describe an essential role for HLA-B-associated transcript 3 (Bat3) in regulating this process. Bat3 co-localizes with DOT1L at histone H3, and Bat3 knockdown results in decreased DOT1L-H3 interaction and H3 K79-diMe, leading to a reduction in IR-induced 53BP1 foci formation. Bat3-depleted cells also exhibit defects in DNA repair and increased sensitivity to IR. We demonstrate that a conserved ubiquitin-like motif of Bat3 and a conserved DOT1L ubiquitin interacting motif are required for DOT1L-Bat3 interaction, which promotes efficient H3 K79-diMe and IR-induced 53BP1 foci formation. Taken together, our data identify a novel role for Bat3 in regulating DOT1L function.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2482. doi:10.1158/1538-7445.AM2011-2482
Abstract 2483: Molecular mechanisms underlying chemopreventive activities of glycyrrhizic acid against UVB-induced carcinogenesis in SKH-1 hairless mouse epidermis
Authors: Cherng Jaw-Ming , Tsai Kuen-Daw , Wei James C. , Perng Daw-Shyong , Yu Alice Y. , Lin Jung-Chung
Published: June 2012

Glycyrrhizic acid (GL) has been shown to possess anti-inflammation, antiviral, and chemoprotective activity against tumors. We assessed the protective effects of GL in UVB-induced skin tumor formation in SKH-1 hairless mice and the early molecular biomarkers. Mice irradiated at 180 mJ/cm2 twice per week elicited 100% tumor incidence in 20 weeks. Feeding of GL prior to UVB irradiation caused a strong protection against photocarcinogenesis in terms of delay in tumor appearance, multiplicity, and size. It took 24 weeks to get 100% tumor incidence in GL plus UVB treated-group; the first tumor appearance in UVB-treated animals occurred at 16th week, which was delayed by 3 weeks in GL plus UVB-treated group. Feeding of GL for 2 weeks before a single UVB (180mJ/cm2) irradiation resulted in a strong and significant decrease in UVB-induced thymine dimer-positive cells (p < 0.004), proliferative cell nuclear antigen PCNA (p < 0.01), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells (p < 0.01) together with an increase in p53 and p21/Cip1-positive cell population in epidermis (p < 0.02 ∼ 0.003). Simultaneously, GL also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and nitric oxide (NO) levels (p < 0.01-0.004). Taken together, these results suggest that GL causes a strong protective effect against photocarcinogenesis via down-regulation of cell proliferative controls, involving thymine dimer, PCNA, apoptosis, transcription factors NF-kB and of inflammatory responses involving COX-2, PGE2, and NO, while up-regulation of p53, p21/Cip1 to prevent DNA damage. These results provide a focus for the rational development of GL as a novel chemopreventive agent against UV-induced human skin cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2483. doi:10.1158/1538-7445.AM2011-2483
Abstract 2487: A multi-kinase inhibitor, XL228, enhanced human cancer cell radiosensitivity and suppressed cell invasion and migration
Authors: Matsumoto Fumihiko , Molkentine David , Clary Douglas O. , Milas Luka , Ang Kian K. , Raju Uma
Published: June 2012

Purpose:, Targeting signaling pathways that are implicated in cancer cell resistance to therapy is being investigated to improve outcome. XL228 is a small molecule inhibitor of IGF-1R, cSrc, BCR/Abl and aurora kinases, which are frequently overexpressed or hyperactive in many cancer types including head and neck squamous cell carcinomas (HNSCCs) and non small cell lung cancers (NSCLCs). This study investigated the potential of XL228 in enhancing cancer cell sensitivity to radiation.

Materials and Methods: Three HNSCC (HN-5, FaDu and UMSCC-1) and three NSCLC lines (H460, A549 and H1299) were used for this in vitro study. Cell viability assay was used to assess the single agent activity of 5-100 nM of XL228 for 1-3 d. In addition, the effects of XL228 (10 or 100 nM) on apoptosis (TUNEL assay), and cell cycle redistribution (flow cytometry), migration and invasion were evaluated. Clonogenic cell survival assayed (in HN-5, FaDu, A549 and H460) after exposing the cells to graded doses of γ-radiation, with or without XL228 (10, 50 or 100 nM) was used to quantify the effect on radiosensitivity.

Results: XL228, at 5-100 nM, reduced survival of all cell lines tested by 10-70% in a dose and time dependent manner and inhibited migration and invasion of two tumors with high propensity to metastasize, FaDu and H460. At 50 and 100 nM XL228 abolished the ability of H460, A549 and FaDu cells to form colony. At 10 nM XL228 significantly increased the radiosensitivity of H460, A549 and FaDu cells by enhancement factors (EF, at the survival fraction of 0.5) of 1.52, 1.31 and 1.67 respectively. But, sensitization of HN-5 cells occurred only at 100 nM (EF = 2.27). Flow cytometry analysis showed that 10 nM of XL228 (that enhanced radiosensitivity of three cell lines) had no effect on the cell cycle distribution and it did not induce apoptosis tested at 24 h after exposure to XL228. However, in HN-5 cells 100 nM of XL228 induced accumulation of cells at the radiation sensitive G2/M phase of the cell cycle and induced apoptosis in 32% of cells. Mechanistic studies including inhibition of DNA repair processes are ongoing.

Conclusions: This study demonstrated that XL228 as a single agent reduced viability, inhibited proliferation, migration and invasion of cells. In combination with radiation, XL228 enhanced HNSCC and NSCLC radiosensitivity. The underlying processes leading to enhanced radiosensitivity may be cell type specific. These data warrant in vivo investigations aiming at taking best regimens to the clinic testing.

(XL228 was provided by Exelixis, Inc. South San Francisco, CA.

Supported by P0-1 CA-06294)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2487. doi:10.1158/1538-7445.AM2011-2487
Abstract 249: Inhibition of protein phosphatase 2A supports androgen-independent growth of prostate cancer cells
Authors: Bhardwaj Arun , Singh Seema , Srivastava Sanjeev K. , Honkanen Richard E. , Reed Eddie , Singh Ajay P.
Published: June 2012

Clinical progression of prostate cancer (PCa) is characterized by a transition from androgen-dependent (AD) to androgen-independent (AI) stage. Once the PCa has recurred in AI form, it progresses to a highly aggressive disease and poses an increased risk of morbidity and death. Therefore, understanding the mechanisms involved in AI progression of PCa is a significant area of research. Earlier, we identified PPP2CA, which encodes for alpha-isoform of the protein phosphatase 2A catalytic subunit (PP2Acα), as one of the downregulated genes in AI PCa cells. PP2A is a ser/thr phosphatase and a potent tumor suppressor involved in broad cellular functions; however, its role in PCa has not yet been determined. Here, we have investigated the effect of PP2A downregulation on the growth of AD PCa (LNCaP) cells under steroid-deprived condition. Furthermore, we have examined the effect of PP2A inhibition on the signaling pathways and delineated their role in AI growth of LNCaP cells. Our data show that the downregulation of PP2A activity by pharmacological inhibition or siRNA-mediated PPP2CA silencing sustains the growth of AD PCa cells under androgen-deprived condition by relieving the androgen-deprivation-induced cell cycle arrest and preventing apoptosis. Immunoblot analysis revealed enhanced phosphorylation of Akt, ERK, BAD, increased expression of cyclins (cyclin A1 and cyclin D1) and decreased expression of cyclin inhibitor (p27) upon PP2A downregulation. Furthermore, our data show that PP2A inhibition partially maintains AR signaling through its increased expression and ligand-independent phosphorylation, which is also supported by AR transcriptional activity assay and its target gene, KLK3, expression. Pharmacological inhibition of Akt, ERK and AR confirmed a role of these signaling pathways in facilitating the AI growth of LNCaP cells. Altogether, our findings suggest that modulation of PP2A activity may represent an alternative therapeutic approach for the treatment of advanced androgen-independent prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 249. doi:10.1158/1538-7445.AM2011-249
Abstract 2496: Inhibition of IL-6 signaling induces radiosensitivity in head and neck squamous cell carcinoma (HNSCC)
Authors: Skinner Heath D. , Su Ying-Wen , Ow Thomas J. , Myers Jeffery
Published: June 2012

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common cancer worldwide. One of the primary modalities used to treat HNSCC is external radiotherapy, however many tumors are resistant to this treatment. Several studies have noted increased expression of IL-6 and IL-6 receptor in a variety of tumors. Furthermore, inhibition of IL-6 using a monoclonal antibody, tocilizumab, as a monotherapy has been shown to decrease tumor growth of HNSCC in pre-clinical models. However the effect of IL-6 signaling on radiosensitivity has not been well characterized. In this study we have found that baseline radioresistance, determined using a standard clonogenic assay, was significantly higher in HNSCC cell lines expressing high levels of IL-6 receptor (IL-6R) compared to those expressing low levels or IL6R as measured by RT-PCR (Surviving fraction at 2Gy (SF2) 65%% vs. 45%, p=0.028). In HN 31 and PCI 15B cell lines, which express high levels of IL-6 receptor, the addition of tocilizumab potentiated radiosensitivity, with a decrease in SF2 of 15% (p<0.05) in both cell lines. This finding was not observed in normal human fibroblasts. Also, the addition of tocilizumab inhibited radiation-induced survival signaling in a dose dependent fashion. Finally, we used immunohistochemistry to show that clinical samples of HNSCC from a variety of sites express IL-6 receptor, compared to little to no staining in surrounding normal tissue. Thus, the addition of tocilizumab to radiotherapy has the potential to enhance the radiosensitivity of HNSCC while minimizing the effect on normal tissue.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2496. doi:10.1158/1538-7445.AM2011-2496
Abstract 2498: Resveratrol administration increases SOD2 and reduces oxidative stress to DNA
Authors: Miller Richard C. , Murley Jeffrey S. , Baker Kenneth L. , Miller Kristin L. , Sullivan Joseph T. , Wardak Hamid , Murley Tyler S. , Grdina David J.
Published: June 2012

Mitochondrial manganese superoxide dismutase (SOD2) activation is a downstream product after stimulation via a variety of signaling pathways from a diversity stimulating agents. Thiols including amifostine (S-2-[3-aminopropylamino]ethylphosphorothioic acid) mesna (sodium-2-mercaptoethanesulfonate), NAC (N-acetyl-L-cysteine) and captopril ([S]-1-[3-mercapto-2-methyl-1-oxo-propyl]-L-proline), are effective inducers of SOD2. Non-thiols including estrogen, cerium oxide, low doses of x-rays, and resveratrol (3,4’,5-trihydroxystilbene) have been shown to induce SOD2 under specific treatment conditions. Studies with resveratrol typically involve continuous exposure of cells for several days before determining the effectiveness of the agent as a radioprotector. In studies reported here, SA-NH murine sarcoma tumor cells established for growth in culture were exposed to a single dose of 50μM resveratrol and analyzed for SOD2 activity from 2hr to 28hr post exposure. Early indications suggest that cells induce SOD2 earlier than typically seen for thiols and other oxidative stress modifiers. Resveratrol increases levels of SOD2 by a factor of 3 compared to controls which is similar to levels observed with thiol exposure. While thiols induce SOD2 via the NFκB pathway, resveratrol acts through the FOXO3a pathway. Studies continue to examine SOD2 activity after exposure to acute exposures resveratrol, amifostine, and x-rays.

This work was supported by DOE grant DE-SC0001271 (D.J.G.) and NIH grant RO1-CA132998 (D.J.G.).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2011-2498
Abstract 2499: Sulfasalazine sensitizes glioblastoma cells to radiation treatment
Authors: Sleire Linda , Wang Jian , Heggdal Jan , Pedersen Paal-Henning , Enger Per Øyvind
Published: June 2012

Glioblastomas (GBMs) are lethal cancers with a limited response to ionizing radiation. Recent studies suggest that Sulfasalazine (SAS), a drug used to treat inflammatory bowel disease, inhibits the xCT antiporter system in glioma cells, thereby blocking their uptake of cystein. Since the availability of cystein is a rate limiting step in intracellular antioxidant production, we wanted to investigate whether sulfasalazine sensitizes glioma cells to radiation.

SAS effect on glioma cell growth was investigated using an electric cell substrate impedance sensing (ECIS) instrument. All cell lines showed altered growth curves in response to SAS treatment. To assess the effect of blocking the xCT antiporter, intracellular levels of the antioxidant glutathione were measured. With increasing doses of SAS, glutathione levels decreased in a dose response manner. Further, U251 glioma cells were treated with escalating doses of SAS, alone or in combination with radiation (8 Gy). Nuclear integrity was evaluated to estimate cell death following treatment. In addition, cell death and viability were investigated using live/dead staining and MTS assay respectively.

All treatment groups exhibited increased rates of cell death compared to untreated controls. However, a combination of SAS and radiation resulted in higher levels of cell death, than radiation or SAS administered alone. In order to assess whether this can be exploited therapeutically, we are currently preparing to treat nude rats harbouring gliblastoma biopsy xenografts with SAS, alone or in combination with radiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2499. doi:10.1158/1538-7445.AM2011-2499
Abstract 252: Evaluating the role of STK33 kinase in mutant KRAS cells
Authors: Babij Carol , Shehabeldin Amro , Fernando Manory , Quon Kim , Kassner Paul , Ruefli-Brasse Astrid , Watson Vivienne , Fajardo Flordeliza , Zhang Yihong , Zondlo James , Sun Yu , Ellison Aaron R. , Plewa Cherylene , Dussault Isabelle , Carnahan Josette
Published: June 2012

Tumors harboring KRAS mutations remain an elusive target for oncology therapeutics. A recent publication (Scholl et al 2009) described a high throughput cellular RNAi screen which suggested a synthetic lethal relationship between STK33 and mutant KRAS. STK33 is a member of the calcium/calmodulin kinase family with a poorly characterized function. Although no genetic alterations in the STK33 gene have been reported in cancer, inhibiting STK33 kinase activity could offer a novel therapeutic approach to target mutant KRAS tumors. Thus, we sought to validate whether the survival of cancer cell lines with mutant KRAS is dependent on STK33, in parallel with a program to identify small molecule inhibitors of STK33 (Zhang Y et al AACR 2011). We focused on mutant KRAS leukemia cells because of their reported dependence on STK33. Using a panel of siRNA, 50-80% knockdown of STK33 at the RNA and protein level was achieved in NOMO-1KRASG13D and SKM-1KRASK117N cells. However we did not observe any alteration in cell viability. In addition, putative STK33 downstream signaling, (phosphorylation of p70 S6K thr389 and RPS6 ser235/236) was unaltered by modulation of STK33 expression. In contrast, knockdown of KRAS caused a significant reduction in both viability and signaling in these cell lines, confirming their dependence on KRAS. A panel of 27 cancer cell lines was screened with an siRNA library representing 1500 druggable genes. STK33 siRNA had no significant effect on viability regardless of KRAS mutational status. As expected, knockdown of KRAS significantly reduced cell viability, especially in mutant KRAS cells. A kinase dead mutant STK33 was used to test the dependence on STK33 kinase activity in mutant KRAS cell lines. Transient over-expression of kinase dead STK33 in PANC-1KRASG12D and DLD-1KRASG13D cells had no effect on viability, nor on the phosphorylation of p70 S6K thr389 and RPS6 ser235/236. In summary we have been unable to confirm a synthetic lethal relationship between STK33 and KRAS. Our data do not support inhibition of STK33 as a promising therapeutic approach for targeting mutant KRAS tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2011-252
Abstract 2524: Targeting microtubule-associated protein 4 with novel small molecule CH4938056 confers antitumor activity under quasi in vivo conditions and in xenograft models
Authors: Kondoh Osamu , Saito Hidemi , Nakanishi Yoshito , Tsukaguchi Toshiyuki , Nagano Kohji , Inomata Noriyuki , Sakata Kiyoaki , Fujii Toshihiko , Satoh Yasuko , Hattori Kazuo , Niizuma Satoshi , Koyama Kohei , Okada Takehiro , Minoshima Yukinori , Kawashima Toshiyuki , Kitamura Toshio , Tsukuda Takuo , Nobuo Shimma , Arisawa Mikio , Ishii Nobuya , Aoki Yuko
Published: June 2012

Tumor microenvironment is a major factor influencing treatment resistance to conventional anticancer therapies. Indeed, under “quasi in vivo” conditions, with low oxygen (1%), low pH (6.5) and low glucose concentration (0.01%) to mimic the environments in grafted tumors in mice or in clinical tumors, anti-proliferative activities of some conventional anticancer agents were diminished. Here, we describe the small molecule CH4938056, which has a novel chemical structure and was identified through cell-based screening under the “quasi in vivo” conditions followed by chemical modification.

Our initial phenotypic profiling revealed that CH4938056 specifically arrests cells at the M phase and that it overcomes multiple resistance mechanisms to conventional anticancer agents including over-expression of MDR1 and BCRP. Then, after designing and identifying a water-soluble phosphate prodrug which successfully converts to CH4938056 after injection, we demonstrated antitumor efficacy of CH4938056 in a HCT116 xenograft model and a MDR1-overexpressing cancer model.

Chemo-proteomic studies and consequent biochemical analysis revealed that CH4938056 binds to microtubule-associated protein 4 (MAP4). Moreover, CH4938056 inhibited MAP4-dependent microtubule assembly in a cell free system. siRNA-mediated knockdown of MAP4 induced chromosomal misalignment in metaphase cells, which closely resembles the primary phenotype of the CH4938056-treated cells. Under the “quasi in vivo” conditions, MAP4 expression turned out to be down-regulated (since MAP4 transcription is known to be negatively regulated by p53 which is up-regulated under these conditions) and when we knocked down MAP4 with siRNA, cancer cells became sensitive to CH4938056, which altogether is consistent with the fact that CH4935056 has antitumor activity even under the “quasi in vivo” conditions. From these observations, we conclude that CH4938056 inhibits proliferation of cancer cells by targeting MAP4. Targeting MAP4 with CH4938056 offers a novel approach for the treatment of cancer, especially for patients resistant to conventional anticancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2011-2524
Abstract 2550: Combination of the KSP inhibitor ARRY-520 with bortezomib causes sustained tumor regressions and significantly increased time to regrowth in bortezomib sensitive and resistant models of multiple myeloma
Authors: Woessner Richard , Tunquist Brian , Walker Duncan , Rana Sumeet , Orlowski Robert , Kuhn Deborah
Published: June 2012

The allosteric kinesin spindle protein (KSP) inhibitor ARRY-520 has potent antitumor activity as a single agent in xenograft models of multiple myeloma, with complete response rates as high as 100%. It has shown promising signs of activity in a phase 1 trial in patients with advanced / refractory multiple myeloma, and a phase 2 study in this patient population is in progress. We report here on the striking activity of ARRY-520 in combination with bortezomib in multiple myeloma xenograft models.

We investigated the activity of the combination of ARRY-520 with bortezomib in several xenograft models of multiple myeloma that are sensitive (Kas-6, ANBL-6, and H929) and resistant (RPMI8226, JJN3, a subline of ANBL-6 made resistant to bortezomib by in vitro treatment, and a subline of RPMI8226 that acquired a PI3K activating mutation) to bortezomib. The combination of ARRY-520 with bortezomib showed additive or superadditive activity, compared to either drug alone, in 6 of the 7 models tested. Complete responses and cures were observed in several models. The Kas-6 model was particularly sensitive, with a 100% cure rate (complete response through day 100). Since previous work from our lab (Mol. Cancer Ther. 9(7), 2046, 2010) implicates MCL1 status as a predictor of response to ARRY-520 in multiple myeloma cell lines, we evaluated the effect of the ARRY-520 + bortezomib combination on MCL1 and other markers of apoptosis. Treatment with the combination resulted in elevation of the cleaved / full length PARP ratio (up to 15X), compared to tumors treated with the single agents, along with a decline in MCL1 levels that coincided with elevation of the level of a 28kd fragment of MCL1 (up to 17X). This shortened form of MCL1 has been reported to be pro-apoptotic. Thus, the cleavage of MCL1 to a proapoptotic form, leading to a decrease in survival signaling and an increase in apoptotic signaling, may play a role in the activity of the ARRY-520 + bortezomib combination. The observation that the combination remained highly active even in models that were poorly responsive to single agent bortezomib suggests that the mechanisms underlying resistance to bortezomib may be distinct from those that contribute to activity of the combination.

These results support ARRY-520 + bortezomib as a rational combination for clinical evaluation, including in patients with bortezomib-refractory disease. A phase 1 trial to evaluate the combination of ARRY-520 + bortezomib + dexamethasone is in progress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2550. doi:10.1158/1538-7445.AM2011-2550
Abstract 2552: Synergistic antitumor effects of FGFR2 inhibitor with 5-fluorouracil on diffuse-type gastric carcinoma
Authors: Yashiro Masakazu , Shinto Osamu , Nakamura Kazunori , Tendo Masashige , Matsuoka Tasuku , Matsuzaki Taro , Kaizaki Ryoji , Miwa Atsushi , Hirakawa Kosei
Published: June 2012

Purpose: Diffuse-type gastric carcinoma carries the highest mortality because of a frequent metastasis to lymph node. S1, a 5-fluorouracil (5-FU) analog, is clinically available for gastric cancer at advanced stage. Fibroblast growth factor receptor 2 (FGFR2) is required for the proliferation of diffuse-type gastric carcinoma. The objective of this study is to clarify the benefit of a combination of S1 and kinase inhibitors including FGFR2 inhibitor Ki23057 in gastric cancer.

Experimental Design: OCUM-2MLN and KATO-III were derived from diffuse-type gastric carcinoma. MKN-7 and MKN-74 were derived from intestinal-type gastric carcinoma. MTT assay was used to examine the growth-inhibitory activity of 5 small-synthetic molecules including Ki23057, Sunitinib, Glivec, Lapatinib, or SU11274, in cells cultured with 5-FU. Combination effects of 5-FU with Ki23057 on proliferation, apoptosis, and mRNA expression were examined. S1 and/or Ki23057 were administered to murine models of diffuse-type gastric carcinoma created by the orthotopic inoculation of OCUM-2MLN cells.

Results: Ki23057 at 100 nM significantly (p<0.01) inhibited the proliferation, and decreased the phosphorylation of FGFR2 in diffuse-type gastric carcinoma cells, but not in intestinal-type gastric carcinoma. Ki23057 showed synergistic anti-tumor effects for diffuse-type gastric carcinoma cells in combination with 5-FU using CalcuSyn analysis, but Sunitinib, Glivec, Lapatinib, and SU11274 did not. The combination of Ki23057 and 5-FU decreased DPD expression, and increased apoptosis rates, and p21 expression level of diffuse-type gastric carcinoma cells. The combined administration of S1 and Ki23057 significantly (p<0.05) decreased orthotopic tumors as well as lymph node metastasis more effectively than S1 alone.

Conclusion: The combined treatment with 5-FU and Ki23057 produced synergistic anti-tumor effects, and is a therapeutically promising for diffuse-type gastric carcinoma treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2552. doi:10.1158/1538-7445.AM2011-2552
Abstract 2573: Combinations of QBI-139, a clinical stage ribonuclease drug
Authors: Strong Laura E. , Kink John A. , Pensinger Daniel , Mei Baigen , Shahan Mark N. , Raines Ronald T.
Published: June 2012

RNA has been recognized as a drug target for cancer therapy, as evidenced by the ongoing clinical trials of RNAi and antisense therapies. An alternative approach that circumvents the delivery and stability issues of RNAi and antisense is to harness the activity of naturally occurring enzymes that degrade RNA. The ribonuclease approach is partially validated by objective clinical responses seen in clinical trials of ribonuclease (RNase) discovered in frog eggs. Our research has focused on creating variants of human RNases with the ability of frog RNase to evade the human RNase inhibitor. Diminished binding of mammalian RNases to the inhibitor have been shown to endow the RNase with the ability to kill cancer cells.

One of the RNase variants, called QBI-139, demonstrated particularly strong anti-cancer activity against a variety of tumor types in in vivo models. QBI-139 is differentiated from the frog RNase because QBI-139 is 95 % identical to a native human RNase, while the frog RNase is only 20% similar. This difference may be what has led to a significantly improved tolerability profile for QBI-139 across species. QBI-139 was selected for clinical development and is now in a Phase I trial of solid tumors.

In charting the clinical development path for QBI-139, the ability of the RNase to work in combination with a variety of standard of care agents had to be evaluated. Based in the single agent activity of QBI-139 in colon, non-small cell lung, ovarian and pancreatic cancers, the potency of QBI-139 in combination with the standard of care agents for these indications was explored.

In in vitro screening, QBI-139 has demonstrated additive and synergistic activity in combination with cisplatin, 5-FU and docetaxel. The treatments were evaluated using fixed ratios of QBI-139 and the combination drug based on their EC50 values. The Combination Index (CI) was determined using median effect analysis. A CI of 1 indicates additive effects, whereas a CI < 1 indicates synergy. Synergy was demonstrated with QBI-139 in combination with cisplatin against solid tumors of non-small cell lung cancer (A549 and H1975) and an additive effect in ovarian (Ovcar-3) cancer. 5-FU and docetaxel also were synergistic with QBI-139 against solid tumors of colon (HCT-116) and ovarian (SK-OV-3) cancer, respectively.The goal of this project is development of an innovative, differentiated therapy, QBI-139, to provide clinical benefit to patients with colon, non-small cell lung and ovarian cancer, which are among the seven deadliest forms of cancer in the United States. In addition to being serious diseases, effective treatment of these cancers also represent unmet medical needs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2573. doi:10.1158/1538-7445.AM2011-2573
Abstract 2576: Mechanism of action of a novel Stat3 inhibitor for cancer therapy
Authors: Buettner Ralf R. , Corzano Renzo , Lin Jianping , Vaidehi Nagarajan , Yip Richard , Chen Yuan , Jove Richard , Horne David , Williams John
Published: June 2012

Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that over-express active Stat3 leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2576. doi:10.1158/1538-7445.AM2011-2576
Abstract 2579: YK5, a small molecule inhibitor of Hsp70 and Hsc70, reveals a multifaceted role for the Hsp70 chaperones in regulating oncogenic and non-oncogenic addiction of tumors
Authors: Rodina Anna , Kang Yanlong , Taldone Tony , Patel Hardik , Patel Pallav , Caldas-Lopes Eloisi , Erdjument-Bromage Hediye , Guzman Monica , Young Jason C. , Melnick Ari , Chiosis Gabriela
Published: June 2012

Background. The cancerous phenotype evolves through accumulation of a large repertoire of elements that allow for increased proliferative potential, self-sufficiency in growth signals and resistance to apoptotic and antiproliferative signals. While much is now gained in understanding the many factors that lead to a malignant phenotype, little remains known on how cancer cells are able to accumulate and to survive with such complexity and high load of aberrant processes.

Study design. Here we use the novel dual Hsp70 and Hsc70 inhibitor, YK5, and its biotinylated derivative, YK55, to investigate the interactome of cytosolic Hsp70s.

Results. We show that heat shock protein 70 family members (Hsp70s) keep active conformations of key oncoproteins and inhibit the function of tumor suppressors. Hsp70s also buffer levels of activated molecules by maintaining them in a conformation that alters their dephosphorylation by phosphatases. Pharmacologic inhibition of Hsp70s by YK5 impairs oncoprotein stability and function, restores tumor suppressor activity, affects major cancer hallmarks and is selectively lethal to cancer cells. We show that enhanced affinity of YK5 for cancer cell-Hsp70s species is an essential mechanism for providing selective tumor lethality.

Conclusion. By demonstrating that pharmacologic dual inhibitors of Hsp70 and Hsc70, such as YK5, confer specific and broad tumor lethality, and interact selectively with tumor Hsp70s species, this work proposes Hsp70s inhibitors as potential anti-cancer therapeutics with a large spectrum of activity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2011-2579
Abstract 2591: Folate receptor alpha: A promoter of cancer cell proliferation that is independent of folate
Authors: Rybinski Katherine A. , Tomkowicz Brian E. , Kamen Barton A. , Grasso Luigi , Nicolaides Nicholas , Sass Philip , Zhou Yuhong
Published: June 2012

Folate receptor alpha (FRα) is over-expressed in malignant tumors of epithelial origin, particularly in ovarian cancer, but largely absent in normal tissue. High levels of protein expression in tumor cells are associated with increased proliferative capacity and enhanced chemo-resistance to Cisplatinum (CP). However, the underlying mechanism remains unclear. We sought to evaluate the biological function of FRα in CP-resistant cancer and hypothesized that alterations of FRα expression levels in drug CP-resistant cancers would result in the re-sensitization of tumor cells to CP. We first developed a human ovarian cancer cell line, IGROV-1, with increased CP resistance and found that these cells also showed an increase in FRα expression over parental IGROV-1 cells. Then, we used FOLR1 specific siRNA to completely knock-down FRα expression. Surprisingly, ablation of FRα expression did not restore sensitivity to CP but had a substantial negative impact on proliferation in both parental and CP-resistant cells. Similar phenomena were also observed when FRα was knocked-down in 786-0 cells, a human renal carcinoma line that expresses robust levels of FRα. In contrast, when we applied FRα knock-down to the normal human kidney cells HK-2 (FRα+), we observed no impairment on proliferation. One explanation of these results is that removal of FRα in cancer cells impairs the ability of these cells to uptake reduced folates, a requirement for nucleic acid synthesis and other methylation reactions. However, even pharmacological concentrations of 5-methyltetrahydrofolate, provided only partial restoration of proliferation in these cells. Taken together, these results suggest that FRα plays an essential role in the proliferation of tumor cells, but not in normal cells via a yet undefined pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2591. doi:10.1158/1538-7445.AM2011-2591
Abstract 2595: KML001 displays vascular-disrupting properties and irinotecan-combined antitumor activities against a murine tumor model
Authors: Min Young Joo , Lee Ho Yong , Moon Chang Hoon , Cha Seung Joo , Lee Hee-Soon , Cha Hee Jeong
Published: June 2012

Purpose: KML001 (sodium metaarsenite) is an orally available arsenic compound that has entered phase II clinical trials in solid tumors. The aim of this study is to assess vascular-disrupting properties of KML001 and to investigate whether KML001 as VDA (vascular-disrupting agent) augments anti-tumor effects of irinotecan in a murine model of colon cancer.

Experimental Design: Female BALBc mice bearing subcutaneos CT26 colon carcinoma cells were injected intraperitoneally with KML001(10 mg/kg) or irinotecan alone, or coadministered weekly for 4 weeks. Moreover, we assessed cytoskeletal changes of HUVEC cells when treated with KML001.

Results: We observed massive necrosis in the central part of the tumor by 24 h after treated with KML001 alone and the regrowth was seen from the periphery of the tumor after 2 days. Otherwise, we observed the significant tumor growth delay when KML001 was administered either 24 hr or 72hr after irinotecan. In addition, we found that KML001 degraded tubulin in HUVEC cells, which might be related with vascular disrupting properties of KML001. Evidence of proteosome-mediated degradation was obtained by demonstrating that adding a proteosome inhibitor.

Conclusions: These results suggest that KML001 is a novel VDA, which can increase anti-tumor activities of irinotecan.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2595. doi:10.1158/1538-7445.AM2011-2595
Abstract 2598: RHAMM- A potential predictor of metastasis and therapeutic target in kidney cancer
Authors: Benitez Anaid , Yates Travis , Chi Andrew , Gahan Jeffrey , Bird Vincent , Lokeshwar Vinata
Published: June 2012

Up to 25% of patients with renal cell carcinoma (RCC) have metastasis at initial diagnosis. Current targeted therapies (e.g., Sorafenib) are not curative and the 5-year survival of these patients is <10%. Molecular determinants of RCC growth and metastasis may be accurate prognostic markers for metastasis and could be targeted for therapy. We evaluated the expression of 5 genes (and their splice variants) as prognostic indicators. We also tested the therapeutic efficacy of the combination of 4-methylumbelliferone (4-MU), a non-toxic, orally bioavailable hyaluronic acid (HA) synthesis inhibitor and Sorafenib (Soraf). Tumor specimens were collected from RCC patients undergoing nephrectomy (n=81; T1, 40; T2+, 41; Grade 1, 5; Grade 2, 32; Grade 3, 25; Grade 4, 19). Nine patients developed metastasis within 24 months. Tissue-RNA was subjected to Q-PCR for HA receptors (CD44, RHAMM), chemokine receptors (CXCR4, CXCR7); and chemokines (SDF1-α,β,γ). mRNA levels were correlated with metastasis by logistic regression. Effect of 4-MU, Soraf and 4-MU+Soraf on cell proliferation, cell cycle and apoptosis was examined in RCC cells (HTB46, 786-O, ACHN, 769-P, A498) by cell counting, flow cytometry and Cell Death ELISA. Boyden chamber was used for motility assay. Effect on cell cycle, apoptosis, and HA receptor levels were evaluated by immunoblotting and Q-PCR. RHAMM expression (157±124) was 8-fold elevated in tumors which later metastasized when compared to those which did not (20.1±31.5; P=0.005). In univariate analysis age, grade, stage and RHAMM levels significantly correlated with metastasis. In multivariate analysis, only RHAMM significantly correlated with metastasis (P=0.03; chi-sq = 4.6; sensitivity 88.9%; specificity 90%). Combination of 4-MU (0.2 mM) and Soraf (2.5 or 5 μM), at non-cytotoxic doses caused 60-90% inhibition in proliferation after 48-72 h treatment and motility in RCC cells. Inhibition of cell proliferation could not be reversed by HA addition. Only the combination induced cycle arrest in late S- and G2-M phases (1.5-2.5-fold) and induced apoptosis (3-4-fold). Cell cycle arrest and apoptosis were confirmed by increased phospho-Chk1 and PARP levels. 4-MU+Soraf combination down regulated CD44 and RHAMM mRNA levels by 2- and 68-fold, respectively. RHAMM expression in RCC tissues is potentially an independent prognostic indicator for metastasis. 4-MU+Soraf combination which targets RHAMM expression inhibits RCC cell growth and motility.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2598. doi:10.1158/1538-7445.AM2011-2598