bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2562: Efficacy of RG7204 (PLX4032), a selective BRAFV600E inhibitor, in combination with pegylated interferon alpha-2a (Pegasys), paclitaxel, carboplatin, and anti-VEGF MAb B20-4.1 in the LOX-IMVI human melanoma xenograft model
Authors: Higgins Brian , Kolinksy Kenneth , Go Zenaida , Char Hing , Lee Richard , Schostack Kathleen , Bollag Gideon , Heimbrook David , Su Fei , Packman Kathryn E.
Published: June 2012

Few drugs are currently approved for the treatment of melanoma, including dacarbazine (chemotherapy) and interferon alpha (immunotherapy). However, only a small fraction of patients with metastatic melanoma respond to dacarbazine and a survival benefit has not been shown with this or any other chemotherapy regimen. Taxanes have demonstrated some activity in melanoma, including modest response rates in combination with carboplatin or bevacizumab. These results contrast with the extraordinary Phase I clinical data reported with the BRAFV600E kinase inhibitor RG7204 (PLX4032), which has demonstrated a remarkable response rate (N Engl J Med 363(9):809-819, 2010). Here we report our preclinical investigation of RG7204 in combination with the biologics interferon alpha-2a (Pegasys) or B20-4.1 (B20) (mouse/ human cross-reactive anti-VEGF MAb), and the chemotherapies paclitaxel and carboplatin in the BRAFV600E mutant LOX-IMVI melanoma model in nude mice.The combination of RG7204 and paclitaxel provided superior survival as compared to either monotherapy. Conversely, the combination of RG7204 plus carboplatin or RG7204 plus B20 did not extend survival as compared to RG7204 monotherapy. When RG7204, paclitaxel, and B20 were given in triplet combination, no improvement in survival was observed over any doublet. Similarly, the triplet combination with RG7204, carboplatin and B20 failed to demonstrate improved survival over any doublets or RG7204 monotherapy. Although complete suppression of tumor growth was readily achieved with either RG7204 or Pegasys monotherapy, the combination provided a remarkable survival benefit with median survival extended by nearly a year as compared to either monotherapy.

Taken together, these data support clinical testing of RG7204 with interferon alpha in melanoma patients harboring BRAFV600E mutation in order to extend survival, and may allow for use of lower dose interferon. Clinical investigation of RG7204 and paclitaxel combination may also be warranted. While our data do not support combined therapy with RG7204 plus anti-VEGF therapy, it should be noted that the LOX model was resistant to B20 monotherapy, and therefore these findings might not apply to tumors with intrinsic sensitivity to anti-VEGF therapy.

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 2562. doi:10.1158/1538-7445.AM2011-2562
Abstract 2569: Combination of the albumin-binding prodrug of doxorubicin (INNO-206) and doxorubicin produces complete remissions in an ovarian A2780 xenograft model
Authors: Kratz Felix , Fichtner Iduna , Graeser Ralph
Published: June 2012

INNO-206, the (6-maleimidocaproyl)hydrazone of doxorubicin (DOXO-EMCH) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that has demonstrated superior antitumor efficacy in eight murine tumor models compared to doxorubicin. After intravenous administration, INNO-206 rapidly binds to the cysteine-34 position of circulating albumin and accumulates in solid tumors due to passive targeting. It has shown objective responses in a phase I study, and phase II studies are planned for 2011 at the recommended dose of 260 mg/m2 i.e. an approximate 4-fold increase over the standard dose for doxorubicin.

Interestingly, in contrast to free doxorubicin, which primarily locates in the nucleus, INNO-206, mainly accumulates in the cytoplasm. Furthermore, the binding of INNO-206 to albumin and the resulting EPR-mediated accumulation and distribution in the tumor will differ from the distribution of free doxorubicin which will be mediated by diffusion. There might thus be a rationale to combine the two drug formulations for obtaining an optimal antitumor effect. Indeed, in cell culture experiments using A2780 cells, a moderate synergy was found at a 5:1 ratio of free Doxo : INNO-206.

In a next step, we therefore tested whether a combination of suboptimal doses of INNO-206 with doxorubicin improved the therapeutic efficacy of INNO-206 or doxorubicin at their respective maximum tolerated doses (MTDs) in the mouse model. We treated nude mice with subcutaneously growing ovarian A2780 xenografts on a weekly schedule with 2 × 8 mg/kg doxorubicin (MTD), 3 × 24 mg/kg INNO-206 (doxorubicin equivalents) (MTD) and a combination of 3 × 12 mg/kg INNO-206 (doxorubicin equivalents) and 3 × 4 mg/kg doxorubicin. In the combination protocol, INNO-206 was administered 6 h prior to doxorubicin treatment. Doxorubicin showed a very moderate effect and a body weight loss of -21 %, whereas INNO-206 and also the combination produced complete remissions. The combination, however, was much better tolerated causing a body weight loss of -12 % compared to -31 % for INNO-206. The surprising result of this experiment is that the doses used in the combination regimen were able to induce complete remission and the best tolerability considering that when dosed individually at these doses, neither doxorubicin or INNO-206 would have produced such an impressive antitumor response. This work is an impetus for investigating further combinations of albumin-binding prodrugs with clinically established anticancer agents.

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 2569. doi:10.1158/1538-7445.AM2011-2569
Abstract 257: Targeted inhibition of mutant k-ras by siRNA in pancreatic cancer cells results in increased sensitivity to anti-EGFR therapy
Authors: Jabbari Neda , Daneker George W. , McDonald John F.
Published: June 2012

Cancer patients are frequently resistant to anti-epidermal growth factor receptor (EGFR) therapy due to secondary mutations in k-ras. This is particularly true for pancreatic cancer patients where the frequency of k-ras mutations is exceptionally high. A variety of therapeutic strategies to target RAS effector pathways have been pursued with limited success. We report here on the use of small interfering RNA (siRNA) to silence oncogenic k-ras in a pancreatic cancer cell line with the goal of sensitizing the cells to anti-EGFR chemotherapies. The AsPC-1 pancreatic cancer cell line expresses EGFR and is homozygous for the mutant k-ras GAT (GGT to GAT in codon 12) allele resulting in the constitutive activation of downstream pathways and reduced apoptotic function. We independently transfected (lipofectamine 2000) AsPC-1 with two siRNAs, one targeted to the mutant (si-k-ras GAT) and one to the wild type (si-k-ras GGT) alleles. We found that levels of k-ras expression (RT-PCR) were significantly reduced after si-k-ras GAT transfection but not after si-k-ras GGT transfection indicating target specificity against the mutant allele. As a preliminary test of the potential clinical utility of si-k-ras GAT mediated inhibition, we measured the relative viability (Tox-8 assay) of AsPC-1 cells transfected with si-k-ras GAT and the EGFR inhibitors Gemcitabine and Erbitux. The results demonstrate a significant increase in sensitivity to both drugs in cells transfected with si-k-ras GAT. We conclude that siRNA mediated inhibition of mutant k-ras may be a promising method by which to sensitize pancreatic cancer cells to anti-EGFR therapy.

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 257. doi:10.1158/1538-7445.AM2011-257
Abstract 2570: Combination studies of EC145 with approved anti-cancer drugs for ovarian cancer
Authors: Reddy Joseph A. , Bloomfield Alicia , Nelson Melissa , Dorton Ryan , Leamon Christopher P.
Published: June 2012

EC145, a potent folate-targeted Vinca alkaloid conjugate, is currently being evaluated in a phase 2 study of EC145 + DOXIL® compared with DOXIL® alone, in women with platinum-resistant ovarian cancer. Since EC145's toxicity profile is appearing to be non-overlapping with many of the existing approved drugs for ovarian cancer, we tested the effects of combining EC145 with some of these drugs. Thus, EC145 was evaluated in combination with cisplatin, carboplatin, docetaxel, topotecan or DOXIL® against folate receptor (FR)-positive human tumor xenograft models in vivo. In these studies, EC145 was purposely administered at a non-curative dose level while the non-targeted combination drugs were given at ∼50-75% of their MTD values. When tested as single agents against the FR-positive KB tumor model, partial anti-tumor responses were typically observed. In contrast, significantly greater anti-tumor activity with no detectable increase in toxicity was observed when EC145 was tested in combination. For example, 100% cures resulted when EC145 was combined with cisplatin or docetaxel, 80% cures with carboplatin or DOXIL®, and 40% cures with topotecan. Overall, these data provide support for the evaluation of EC145 in combination with numerous agents with different mechanisms of action for the treatment of 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 2570. doi:10.1158/1538-7445.AM2011-2570
Abstract 2571: ABT-737, a small molecule Bcl-2/Bcl-xL antagonist, increases docetaxel-mediated apoptosis in human prostate cancer cells
Authors: Parrondo Ricardo , de las Pozas Alicia , Reiner Teresita , Perez-Stable Carlos
Published: June 2012

Castration-resistant prostate cancer (CRPC) express high levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL, resulting in drug resistance and association with poor prognosis. Docetaxel (Doc), an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit but patients eventually become resistant to Doc with little hope for survival. It is apparent that Doc chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing pro-apoptotic signals when combined with other drugs. We investigated whether ABT-737 can increase Doc-mediated apoptosis in CRPC cells. Our results in androgen-dependent LNCaP and CRPC PC3 cell lines indicate that ABT-737 (1 μM) increases Doc (1 nM)-mediated cell death and caspase-dependent apoptosis, as determined by trypan blue exclusion, annexin-FITC/propidium iodide flow cytometry, and Western blot analysis. Our data suggests that Doc's anti-cancer effect results from prolonged cyclin B1/Cdk1 activation and inactivation of Bcl-2/Bcl-xL by phosphorylation, resulting in increased apoptosis. We show a correlation between high phosphorylation (P) of Bcl-2(Ser70)/Bcl-xL(Ser62) and increased apoptosis by Doc and Doc + ABT-737 treatments. Furthermore, the Cdk1 inhibitor purvalanol A blocks Doc + ABT-737-mediated increases in P-Bcl-2/P-Bcl-xL and subsequent apoptotic cell death. Lentivirus pLKO.1 shRNA stable knockdown of Bax but not Bak lowers cell death LNCaP and PC3 cells after treatment with Doc + ABT-737. In contrast to LNCaP and PC3, the CRPC DU145 cell line is null for Bax, expresses high levels of anti-apoptotic Mcl-1, and is more resistant to ABT-737. Retrovirus pBABE stable expression of Bax in DU145 cells restores sensitivity to Doc + ABT-737 compared to the empty vector control cells. We conclude that ABT-737 can sensitize CRPC cells to Doc treatment and may provide an important combination chemotherapy strategy for improving overall survival.

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 2571. doi:10.1158/1538-7445.AM2011-2571
Abstract 258: The role of EGFR on RAF inhibitor resistance in B-RAFV600E melanoma and colon tumors
Authors: Yen Ivana , Sideris Steve , Song Kyung , Liu Bonnie , Schmidt Stephen , Koeppen Hartmut , Belvin Marcia , Friedman Lori , Hatzivassiliou Georgia , Malek Shiva
Published: June 2012

Constitutive activation of the RAS, RAF, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) pathway is a common finding in many human cancers. Activating mutations in K-RAS or B-RAF constitute over 30% of all mutations in human tumors. It has been demonstrated that RAF inhibitors selectively inhibit B-RAFV600E tumors and not RAS mutant tumors, despite functioning as one of the key effector enzymes downstream of RAS and upstream of MEK. Recent Phase I clinical data with RAF inhibitors has demonstrated a significant response rate (81% response rate) in metastatic melanoma patients with B-RAFV600E-positive tumors (N Engl J Med. 2010 363:809-19). In contrast, the response rates in B-RAFV600E-positive colon tumors have been relatively low. In this work we examine the molecular mechanism of EGFR pathway activation as a mechanism of resistance by comparing the effects of RAF inhibitors against a panel of B-RAFV600E, mutant K-RAS, and WT RAS/RAF tumor lines in the presence and absence of EGF stimulation. We demonstrate that in B-RAF WT lines, pathway induction is observed for RAF inhibitors as evidenced by increases in phospho-MEK and phospho-ERK levels. RAF-inhibitor induced pathway activation in this setting mimics the kinetics and mechanism of EGF-induced pathway stimulation where RAS-GTP levels, C-RAF kinase activity and pERK levels are elevated. Upon stimulation with EGF or serum in B-RAF WT or K-RAS mutant lines, RAF inhibitor induced pathway activation is attenuated. In contrast in B-RAFV600E tumor lines, RAF inhibitors effectively block the MAPK pathway under basal conditions but become ineffective in a subset of B-RAFV600E lines when cells are stimulated with EGF. Additional studies across a broader panel of colon and melanoma mutant B-RAF lines demonstrate that B-RAFV600E lines with high basal levels of EGFR tend to be more resistant to selective B-RAFV600E inhibitors. Immunohistochemistry of colorectal tumor patient samples with either wildtype B-RAF or B-RAFV600E illustrates that colorectal B-RAFV600E tumors are associated with a higher percentage of membrane EGFR. Taken together, these results provide insight into the therapeutic utility of MAPK pathway inhibitors, and emphasize not only the importance of targeting defined genetic backgrounds in cancer treatment but the value in assessing other key pathway markers (such as EGFR) in B-RAFV600E-positive tumor subsets as a factor in sensitivity or resistance to therapy.

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 258. doi:10.1158/1538-7445.AM2011-258
Abstract 2593: A novel small molecule drug (LWKS) for pancreatic cancer therapy
Authors: Wang Yan , Maeda Yoshiko , Qin Sheng , Lam Kit , Liu Ruiwu , Kumaresan Pappanaicken R.
Published: June 2012

Pancreatic ductal adenocarcinoma (PDAC) currently stands at approximately 29,000 new cases per year in North America and lack of effective therapies leads to an extremely poor prognosis. Since pancreatic cancers respond poorly to chemotherapy, more effective and less toxic treatment is a crucial need. We have employed a biological assay (MTS assay) to identify small molecular compounds that induce cytotoxicity specifically in pancreatic cancer cells and not in normal cells by screening large number of compounds from various combinatorial chemical library. Commercially available PDAC cells derived from various stages of pancreatic cancer were used for screening. For normal cells, immortalized epithelial cells isolated from pancreatic duct (h-tert HPNE) were used. One of the compounds, LWKS (MW: 547.94) kills pancreatic cancer cells and not the normal HPNE cells. LWKS kills pancreatic cancer cells isolated from all the four stages and the EC50 is in the range of 2 – 10 µM, whereas for normal cells is 70 µM. Maximum tolerated dose is 80 mg/kg of body weight. At 20 mg/Kg body weight, it showed significant tumor shrinkage in human PDAC xenografts mice model when compared to PBS controls. The tumor reduction was monitored by measuring the tumor volume for two times per week for 8 weeks. All the animal safety procedures were followed as described by AALAC. At this dosage level no toxicity was observed in the mice model.

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 2593. doi:10.1158/1538-7445.AM2011-2593
Abstract 2611: Inhibition of the immunoproteasome subunit LMP2 by UK-101 leads to apoptosis and cell cycle arrest in pancreatic cancer
Authors: Jang Eun Ryoung , Thakkar Nilay , Kim Kyunghwa , Lee Eun Y. , Kim Kyung-Bo , Lee Wooin
Published: June 2012

Background: The immunoproteasome is an alternative form of the constitutive proteasome and it has been increasingly shown to play an important role in a number of diseases including cancer and inflammatory diseases. We have previously developed ‘UK-101’, a small molecule that selectively inhibits the immunoproteasome catalytic subunit LMP2. In our present study, we investigated the therapeutic potential of the immunoproteasome-targeting approach in pancreatic cancer using UK-101 as a lead compound.

Methods: The expression of LMP2 was assessed in primary pancreatic cancer tissues and established pancreatic cancer cell lines via immunohistochemical (IHC) and immunoblotting analyses. Using pancreatic cancer cells expressing LMP2, we have investigated the effects of UK-101 (an LMP2 inhibitor), general proteasome inhibitors (bortezomib and carfilzomib) and gemcitabine on cell survival, apoptosis and cell cycle progression. We also examined the effects of UK-101 on various molecular markers associated with apoptosis and cell cycle control.

Results: Our immunoblotting and IHC analyses indicated that the LMP2 levels are substantially upregulated in primary pancreatic cancer tissues as well as established pancreatic cancer cell lines compared to nonmalignant pancreatic tissues. The IHC staining on a tumor tissue array demonstrated positive immunostaining for LMP2 in approximately 53% of pancreatic cancer specimens evaluated (n=23 out of 43) while the normal pancreatic duct showed a negative LMP2 staining (n=11). Using Panc-1 (gemcitabine-resistant) and BxPc-3 (gemcitabine-sensitive) cells, we observed that the UK-101 treatment leads to a covalent modification of LMP2, a decreased proteasomal proteolytic activity as well as apoptotic cell death and cell cycle arrest.

Conclusion: Our results show that LMP2 is upregulated in pancreatic cancer and that the targeted inhibition of LMP2 using UK-101 induces apoptotic cell death and cell cycle arrest. Taken together, these results suggest that the immunoproteasome may be a potential therapeutic target that can be exploited for pancreatic cancer where the existing therapy including gemcitabine shows limited efficacy.

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 2611. doi:10.1158/1538-7445.AM2011-2611
Abstract 2641: Diversity of anticancer mechanisms of action of tritepenoids derived from betulinic acid
Authors: Dzubak Petr , Rehulka Jiri , Holub Dusan , Ozdian Tomas , Janostakova Anna , Sarek Jan , Vlk Martin , Hajduch Marian
Published: June 2012

Betulinic acid and its derivatives are cytotoxic compounds with the specific effect to the tumor cells. In our betulinic acid derivative chemical library we have identified several potent compounds which were highly active in the wide spectrum of the cancer cell lines and the primary tumour cells as well as. Cell cycle modulating effects of selected compounds were monitored and surprisingly the compounds with different effect on the cell cycle profile were observed. Some derivatives were inhibiting the progression through the early S or G2-M phases and some were rapidly inducing the apoptosis. To identify the mechanisms of the action we have used genomic expressional (Affymetrix) and proteomic profiling (LC-MS/MS, SILAC). Derivatives inducing the rapid induction of the apoptosis were identified to regulate and affect the mRNA expression of the genes and proteins closely related to the oxidative stress and to the detoxification enzymes. Glutathione detoxification pathway and the oxidative phosphorylation cascade (upregulation of glutathion peroxidase, peroxiredoxine, thioredoxin, sulfiredoxine, cytochrome c oxidase, down regulation of glutathion S transferase) were affected and direct interaction of derivatives with cytochrome c was observed. All previously mentioned mechanisms are closely related to the mitochondrion damage. However, derivatives inducing the G2/M block were also increasing the expression of the tubulin chaperone cofactor A (TBCA) and the underexpression of other proteins like chaperonin containing TCP1, subunit 3 (CCT3) related to tubulin polymerization pathway and cell cycle regulation. Tubulin chaperone cofactors were recently suggested to be potential targets for the cancer therapy and current works are showing the upregulation of tubulin chaperone cofactor C correlated to the suppression of the tumor growth and increased chemosensitivity in resistant cancer phenotypes. This work was supported by the GACR 301/09/P433, GACR 305/09/1216 and CZ.1.05/2.1.00/01.0030).

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 2641. doi:10.1158/1538-7445.AM2011-2641
Abstract 2643: Inhibition of calpain disrupts the autophagic response initiated by bortezomib resulting in increased death of myeloma cells
Authors: Landowski Terry H. , Escalante Aluvia M. , McGrath Ryan , Karolak Matthew R. , Henderson Meredith , Perrian Georgia O. , Lynch Ron
Published: June 2012

The proteasome is a multi-subunit enzyme complex that plays a central role in the regulation of proteins controlling many important cellular functions including cell cycle progression, differentiation, gene transcription and apoptosis. Bortezomib is a potent proteasome inhibitor that has recently been approved for use in refractory multiple myeloma. Our previous work demonstrated that bortezomib induces an endoplasmic reticulum (ER) stress response ultimately leading to Ca2+-dependent apoptotic cell death. Intracellular Ca2+ has also been implicated in the cellular stress response known as autophagy. The biochemical events linking the cellular stress response with the induction of autophagy, and the relationship between autophagy and apoptosis are not well understood.

In this study, we investigate the role of the Ca2+ dependent serine protease, calpain, as a mediator of the conversion from autophagic cell survival to accelerated cell death in the ER stress response. Using live cell imaging we demonstrate that bortezomib rapidly initiates autophagy in myeloma cells. Microtubule associated protein 1 light chain 3 beta (LC3) is an autophagic marker that is converted from the inactive form (LC3I) to the autophagosomal membrane-associated (LC3I) form. Simultaneous analysis of the LC3II/I ratio and the polyubiquitin-binding protein, p62/SQSTM1, demonstrates an early elevation in autophagosome formation with an increase in autophagic digestion. Inhibition of the Ca2+-dependent serine protease, calpain, dramatically increases the accumulation of non-functional autophagosomes illustrated by a rise in both the LC3II/I ratio and p62 levels. This effect is accompanied by a significant enhancement or acceleration of cell death. Similarly, elimination of the small catalytic subunit, CAPNS1 using siRNA significantly increased bortezomib-mediated cell death. Our data suggest that inhibition of calpain prevents autophagolysosome maturation, and subverts the survival response thereby enhancing cell death. These data suggest a new therapeutic strategy to enhance the activity of proteasome inhibitors and overcome drug resistance in refractory myeloma and other B cell malignancies.

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 2643. doi:10.1158/1538-7445.AM2011-2643
Abstract 2663: Dual cytoprotective and cytotoxic functions of autophagy in the response ofbreast tumor cells to radiation and in radiosensitization by vitamin D3
Authors: Bristol Molly L. , Di Xu , Gewirtz David A.
Published: June 2012

Introduction: While chemotherapy and radiation treatment may be initially effective at suppressing breast cancer growth, disease recurrence and tumor metastasis are major clinical problems. A number of strategies have been developed in an attempt to enhance the response to radiation and prevent tumor cell recovery by interfering with cytoprotective signaling. Autophagy, a cellular process in which the degradation of subcellular organelles generates energy and metabolic precursors, may be cytoprotective or cytotoxic, depending on the cells and the nature of the stressful challenge. Our studies were designed to evaluate the involvement of autophagy in radiosensitization by the hormonally active form of vitamin D3, 1,25D3. Additionally, studies were designed to test the premise that interference with autophagy would influence sensitivity to treatment with 1,25D3 + radiation.

Results: Our studies demonstrate radiosensitization of breast tumor cells by vitamin D (1,25D3) through the promotion of autophagy as well as a concurrent cytoprotective function of autophagy in irradiated cells. 1,25D3 promoted autophagy (as well as autophagic flux) in irradiated MCF7 cells and suppressed the proliferative recovery that occurs after radiation alone. 1,25D3 also enhanced radiosensitivity and promoted autophagy in p53 mutant Hs578t breast tumor cells as well as MCF7 cells that overexpress Her-2/neu. In contrast, 1,25D3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D3 was not attenuated by either a pharmacological or genetic block to autophagy due to a shift in the mode of cell death to apoptosis. In contrast to autophagy mediating the cytotoxic impact of the 1,25D3 and radiation combination treatment, radiation alone promoted a low level of autophagy that was cytoprotective. Specifically, pharmacological or genetic interference with autophagy induced by radiation resulted in cell killing (primarily by apoptosis). These studies demonstrate that in the same experimental system, autophagy can function both as a mechanism for evading cell killing by radiation and as a pathway for mediating the killing of breast tumor cells by radiation.

Conclusions: Both the suppression of cytoprotective autophagy and the promotion of cytotoxic autophagy represent potential strategies for radiation sensitization in breast cancer.

Supported, in part, by American Institute for Cancer Research Grant # 06A058-REV and by DOD predoctoral training grant W81XWH-09-1-0020.

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 2663. doi:10.1158/1538-7445.AM2011-2663
Abstract 2666: Relationship between chromatin structure and sensitivity to molecularly-targeted radiation therapy
Authors: Terry Samantha Y.A , Vallis Katherine A.
Published: June 2012

The open structure of euchromatin renders it more susceptible to DNA damage by ionising radiation (IR) compared to compact heterochromatin. The goal was to investigate whether chromatin accessibility also affects the toxicity of Auger electron-emitting radiopharmaceuticals.

SAHA is a histone deacetylase inhibitor that results in chromatin remodelling to an open structure and is used to treat cutaneous T-cell lymphoma. Epidermal growth factor receptor (EGFR)-overexpressing MDA-MB-468 and MDA-MB-231/H2N breast cancer cells were treated with SAHA and irradiated (137Cs source, 1Gy/min, 0.5Gy) or incubated with 111Indium-DTPA-hEGF (111In-EGF) for one hour. Cells were stained for γH2AX, a marker of DNA double-strand breaks, 30min after γ-irradiation or after exposure to 111In-EGF. Nuclear uptake of 111In following SAHA was evaluated in cellular fractionation experiments. Total cellular H2AX protein expression after SAHA treatment was analysed by western blot. Cells treated with SAHA and consequently irradiated (up to 6Gy) or incubated with 111In-EGF (24h), were plated and incubated for 7-10 days in clonogenic survival experiments. Cells were exposed to calyculin A (50nM) or NaCl (0.5M), which result in chromatin condensation, irradiated (1Gy) or incubated with 111In-EGF (1h) and stained for γH2AX as before.

In both MDA-MB-468 and MDA-MB-231/H2N cells, SAHA resulted in an increase in the number of γH2AX foci (16 and 14 per cell respectively) compared to IR alone (12 and 11 foci/cell respectively). SAHA plus 111In-EGF also increased DNA damage (22 and 12 foci/cell respectively) compared to 111In-EGF alone (15 and 7 foci/cell respectively). SAHA did not alter the extent of nuclear uptake of 111In in either cell line (0.4-0.6% of total administered), nor did it alter total cellular H2AX protein expression. Preliminary clonogenic data suggest that SAHA decreases cell survival after either irradiation or incubation with 111In-EGF in both cell lines. MDA-MB-468 cells treated with calyculin A accumulated less damage after γ-irradiation (11 foci/cell) compared to IR alone (20 foci/cell). Treatment of both cell lines with NaCl before irradiation (11 and 9 foci/cell) also protected against DNA damage compared to IR alone (19 and 16 foci/cell). Preliminary data suggests that cells treated with NaCl prior to incubation with 111In-EGF also acquired less DNA damage.

Chromatin density modification alters radiosensitivity and DNA damage after γ-irradiation. The results of this study indicate that this is also the case for Auger electron-emitting molecularly-targeted radiation therapeutic agents.

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 2666. doi:10.1158/1538-7445.AM2011-2666
Abstract 2677: Novel Hsp90 inhibitor, STA-9090, for combination with radiotherapy
Authors: Sang Jim , Smith Donald , Korbut Tim , Zhang Chaohua , Proia David A. , Wada Yumiko
Published: June 2012

Introduction: Radiation is accepted as an important standard therapy for locally unresectable cancers, and as such is given to approximately 60% of cancer patients. However, radio-resistance and repair of sublethal radiation damage can limit its efficacy. Recent studies have shown that Heat Shock Protein 90 (Hsp90), a molecular chaperone that mediates maturation and activation of client proteins, plays a critical role in establishing resistance to radiation therapy. Inhibiting Hsp90 has been reported to sensitize tumors to radiation, resulting in tumor growth suppression and augmenting therapeutic cell death induction. Unfortunately, many of the Hsp90 inhibitors currently in clinical trials exhibit hepatotoxicity as well as ocular toxicity, hindering their clinical use. Taken together, development of clinically acceptable Hsp90 inhibitors for combination with radiation could serve as an important strategy for improving radiotherapy success.

STA-9090 is a second generation Hsp90 inhibitor that has shown potent preclinical activity and is currently in twelve Phase II trials across a broad range of indications. STA-9090 has demonstrated encouraging activity in a Phase II trial in patients with stage IIIB and IV non-small cell lung cancer. Importantly, STA-9090 has displayed a favorable safety profile with substantially lower incidence of hepatic or ocular toxicity than that reported for other Hsp90 inhibitors.

Results: We evaluated the radiosensitizing potential of STA-9090 in vivo. Monotherapy treatment with either STA-9090 or 2 Gray (Gy) ionizing irradiation resulted in moderate reductions in human tumor growth rates in a mouse xenograft model. Combination of STA-9090 with 2 Gy irradiation resulted in substantial tumor regression. Increasing the dose of radiation in the combination arm to 4 Gy further enhanced tumor regression, resulting in a 50% reduction in tumor volume. In summary, STA-9090 offers a safe and effective strategy for improving the outcome of radiotherapy in human 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 2677. doi:10.1158/1538-7445.AM2011-2677
Abstract 2679: Gold nanoparticles enhance the radiation therapy of a murine squamous cell carcinoma
Authors: Hainfeld James F. , Dilmanian Avraham , Zhong Zhong , Slatkin Daniel N. , Kalef-Ezra John A. , Smilowitz Henry M.
Published: June 2012

The purpose of this study is to test the hypothesis that gold nanoparticle (AuNP, nanogold)-enhanced radiation therapy (nanogold radiation therapy, NRT) is efficacious when treating the radiation resistant and highly aggressive mouse head and neck squamous cell carcinoma model, SCCVII, and to identify parameters influencing the efficacy of NRT. Subcutaneous (sc) SCCVII leg tumors in mice were irradiated with x-rays at the Brookhaven National Laboratory (BNL) National Synchrotron Light Source (NSLS) with and without prior intravenous (iv) administration of AuNPs. Variables studied included radiation dose, beam energy, temporal fractionation and hyperthermia. AuNP-mediated NRT was shown to be effective for the sc SCCVII model. AuNPs were more effective at 42 Gy than at 30 Gy (both at 68 keV median beam energy) compared to controls without gold. Similarly, at 157 keV median beam energy, 50.6 Gy NRT was more effective than 44 Gy NRT. At the same radiation dose (∼42 Gy), 68 keV was more effective than 157 keV. Hyperthermia and radiation therapy (RT) were synergistic and AuNPs enhanced this synergy, thereby further reducing TCD50 s (tumor control dose 50%) and increasing long-term survivals. It is concluded that gold nanoparticles enhance the radiation therapy of a radioresistant mouse squamous cell carcinoma. The data show that radiation dose, energy and hyperthermia influence efficacy and better define the potential utility of gold nanoparticles for cancer x-ray therapy.

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 2679. doi:10.1158/1538-7445.AM2011-2679
Abstract 2680: Characterization of tumor associated antigens using a recombinant expression of a patient derived breast cancer cDNA library and autologous serum using the SEREX technique
Authors: Novinger Leah J. , Shukla Girja , Pero Stephanie , Krag David N.
Published: June 2012

Autoantibodies to tumor proteins in patient serum may serve as biomarkers in breast cancer diagnosis or as a potential targeted therapeutic when conjugated to drugs or radiotherapies. The tumor associated antigens (TAAs) to which they bind may be of further diagnostic or prognostic value. In order to identify TAAs, a cDNA library was created from a patient breast tumor, recombinantly expressed with lambda phage in XL1-Blue cells, and tested against autologous patient serum to identify potential TAAs using a modified technique based on serological analysis of recombinant cDNA expression libraries (SEREX) that allows for much smaller quantities of source material and reduces library production time. Identified TAAs and serum autoantibody concentrations against those proteins will be presented in the context of developing a new customized therapy for patients with breast 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 2680. doi:10.1158/1538-7445.AM2011-2680
Abstract 2689: XAGE-1b(GAGED2a) immunity in non-small cell lung cancer
Authors: Ohue Yoshihiro , Eikawa Shingo , Matsumoto Hirofumi , Mizote Yu , Isobe Midori , Uenaka Akiko , Fukuda Minoru , Nakayama Eiichi , Oka Mikio
Published: June 2012

BACKGROUND: Several cancer/testis(CT) antigens such as NY-ESO-1 antigen, etc., have been shown to elicit immune responses in cancer patients spontaneously. Because of their restricted expression in normal tissues and high immunogenicity, CT antigens have been thought to be attractive targets for cancer vaccine. We previously described XAGE-1b (GAGED2a), one of the CT-associated antigens, was predominantly expressed in lung adenocarcinoma by immunohistochemistry using XAGE-1b mAb (clone USO 9-13) and could elicit both humoral and cellular immune response in patients with non-small cell lung cancer (NSCLC). In this study, we established CD4 and CD8 T cell clones from PBMC in XAGE-1b antibody-positive patients and analyzed the antigen recognition.

MATERIALS AND METHODS: Sera and PBMCs were obtained from NSCLC patients who visited Kawasaki Medical School Hospital between 2005 and 2010. Antibody response to XAGE-1b was analyzed by ELISA using synthesized XAGE-1b protein. CD4 and CD8 T cell responses against XAGE-1b were examined by IFN-γ ELISA and/or capture assay using 17 16- or 17-mer XAGE-1b-overlapping peptides (OLPs) spanning the entire XAGE-1b protein by FACS in antibody-positive patients. Cytotoxicity was analyzed by bioluminescence assay (GAPDH).

RESULTS: Spontaneous CD4 and CD8 T cell responses were also detected in most of the antibody positive patients. Occurrence of antibody response, and CD4 and CD8 T cell responses in XAGE-1b (GAGED2a) antibody positive patients indicated strong immunogenicity of XAGE-1b (GAGED2a) antigen in NSCLC patients. Furthermore, We identified the minimal epitope peptide bound to HLA-DR4.1 and DP5 recognized by CD4 and those bound to HLA-A2, B61 and Cw1 recognized by CD8 T cell clones. CD4 and CD8 T cell clones recognized naturally processed XAGE-1b (GAGED2a) antigen on APC (antigen-presenting cell) and the CD8 T cell clone was stained with the peptide/MHCI tetramer and showed cytotoxicity against XAGE-1b (GAGED2a)-expressing, appropriate HLA class I expressing tumor cell line.

CONCLUSION: Our findings indicate validity of XAGE-1b (GAGED2a) for cancer vaccine. Now we plan to global clinical trial of cancer vaccine against XAGE-1b in NSCLC.

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 2689. doi:10.1158/1538-7445.AM2011-2689
Abstract 2691: Regulation of ultraviolet radiation induced cutaneous tumor development by Toll like receptor-4
Authors: McConnell Matthew , Yusuf Nabiha
Published: June 2012

Ultraviolet (UV) B radiation, in particular the mid-wavelength range (UVB; 290-320 nm), is one of the most significant risk factors for the development of skin cancer. Toll like receptor-4 (TLR4) has been shown to play an important role in cancer, but the role that it plays apparently depends on the stage of tumor development, the tumor type and the initiating agent. The purpose of this study was to delineate the contribution of TLR4 in UVB induced cutaneous tumor development using TLR4 deficient C3H/HeJ mice and TLR4 proficient C3H/HeN (WT) mice. We observed that UVB induced skin carcinogenesis was retarded in terms of tumor incidence, and tumor latency, in mice deficient in TLR4 compared to WT mice, whereas significantly greater (p<0.05) numbers of tumors occurred in WT mice. Further, we found that tumor-bearing WT mice produced more IL-17 and IL-10 (p<0.05), whereas TLR4 deficient mice produced more IFN-γ (p<0.05). CD4+CD25+ regulatory T-cells from WT mice produced more IL-10 (p<0.05) than regulatory T-cells from TLR4 deficient mice. There was a significant increase (p<0.05) in Foxp3 expression in skin of WT mice than TLR4 deficient mice. Time course kinetics of tumor development revealed an increase in MyD88 and TRAF6 expression in skin and tumors of WT mice in comparison to TLR4 deficient mice. These results indicate that, TLR4 promotes tumor development in this model. Strategies to inhibit TLR4 may allow us to develop immunopreventive and immunotherapeutic approaches for management of UVB induced skin 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 2691. doi:10.1158/1538-7445.AM2011-2691
Abstract 2693: Amplification of tumor immunity with cryoablation
Authors: Veenstra Jesse , Littrup Peter , Wei Wei-zen
Published: June 2012

Tumor cryoablation, the use of freezing temperatures to destroy tumor tissue while preserving tumor-associated antigens, has the potential to initiate or amplify tumor immunity. However, enhanced tumor immunity is not consistently observed in treated patients, suggesting inadequate immune priming by cryoablation itself. We hypothesize that cryoablation in coordination with DNA vaccination and/or immune modulation will amplify effector mechanisms to provide long-term anti-tumor protection. BALB/c mice implanted with a neu expressing mammary tumor TUBO received two electro-vaccinations 10 days apart, with neu DNA or blank vector, followed 4 days later with cryoablation or sham surgery of the tumor. Mice receiving both neu vaccination and cryoablation produced a 3-fold higher anti-neu antibody titer over neu vaccination alone (P<0.02) and a 16-fold higher titer over cryoablation alone (P<0.003). Combination treatment also provided full protection against TUBO re-challenge, supporting enhanced immunity by cryoablation. To test the effect of combining cryoablation with the depletion of regulatory T cells (Treg), 12 week old BALB Neu transgenic mice which had spontaneously developed ductal carcinoma in situ in the mammary glands were treated with α-CD25 mAb to deplete Tregs, partial cryoablation of the 4th mammary gland, or a combination of both. Development of palpable mammary tumor throughout all ten mammary glands was significantly delayed in mice receiving combination treatment over either treatment alone, indicating amplification of tumor immunity in Treg depleted mice by cryoablation. The mechanisms of amplified immunity are being investigated by analyzing a panel of inflammatory cytokines in the serum as well as gene expression in lymphocytes in the tumor, spleen, and tumor draining lymph nodes at different points after cryoablation. Phenotyping of tumor infiltrating immune cells post-cryoablation is also being assessed.

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 2693. doi:10.1158/1538-7445.AM2011-2693
Abstract 2702: Sorafenib modulates immune responses in patients with hepatocellular carcinoma
Authors: Cabrera Roniel , Ararat Miguel , Cao Mengde , Xu Yiling , Wasserfall Clive , Brusko Todd , Atkinson Mark A. , Liu Chen , Nelson David R.
Published: June 2012

Background/Aim: We have previously shown that patients with hepatocellular carcinoma (HCC) have impaired immunity. The poor immune reactivity is marked by blunted effector CD4 T cell responses (Teff), elevations in immunosuppressive soluble factors, and an increase of CD4+CD25+ regulatory T cells (Tregs) with enhanced suppression. Presently, sorafenib is the only systemic agent with a proven survival benefit for unresectable HCC. We hypothesize that sorafenib has immune modulatory properties that may partially explain the clinical benefit. In this study, we characterize the effects of sorafenib on the function of CD4 Teffs and Tregs.

Methods: PBMC cells isolated from normal healthy controls (NHC), disease controls (DC) and HCC patients were used to isolate CD4+CD25- Teffs and Tregs to use in functional immune assays. The 3H thymidime incorporation and the IFNg ELISpot were used to assess CD4 Teff responses.

Results: Sorafenib increased Teff proliferation in HCC patients during dose response cultures. We observed a similar positive effect by sorafenib on Teff IFNg production in HCC patients. The dose related effect of sorafenib on Teff proliferation and INFg production occurred in all three groups – HCC, DC, and NHC. However, Teff responses in HCC patients improved with lower concentrations of sorafenib (0.1uM) while the Teff responses from DC/NHC increased only after the introduction of higher concentrations of sorafenib (0.7uM). Further, while sorafenib improved Teff immune responsiveness in HCC patients in a dose dependent manner (0.1uM to 2 uM), the increase in Teff responsiveness in DC/NHC peaked and leveled off at lower concentrations (0.7 uM). However, sorafenib was unable to abrogate the suppressive properties of autologous HCC derived serum on Teff responses at the dose range studied. Interestingly, sorafenib decreased Treg mediated suppression during suppression assays using Treg:Teff co-cultures at a 1:1 ratio.

Conclusion: Sorafenib appears to have a positive modulatory effect on immune responses by enhancing CD4 Teff function and decreasing Treg suppressive function in HCC patients. Studies that investigate new approaches that combine sorafenib with other agents that target inhibitory immune pathways are needed.

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 2702. doi:10.1158/1538-7445.AM2011-2702