bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1664: Maternal embryonic leucine aipper kinase (MELK) is a critical regulator of proliferation and is independently prognostic in estrogen receptor-negative breast cancer
Authors: Wheatley Karrie , Speers Corey , Tsimelzon Anna , Sexton Krystal , Hilsenbeck Susan , Chang Jenny , Brown Powel
Published: June 2012

Background: Molecularly targeted therapies such as tamoxifen, aromatase inhibitors, and trastuzumab are useful for the treatment of breast cancer. However, these targeted therapies do not treat all breast cancers, especially estrogen receptor alpha (ER)-negative, progesterone receptor (PR)-negative, HER2-negative (“triple-negative”) breast cancer. Our lab previously used gene expression profiling of human breast cancers to identify kinases overexpressed in ER-negative breast cancers. One of the kinases highly expressed in a poor prognosis group of triple negative breast cancers is maternal embryonic leucine-zipper kinase (MELK). The purpose of this study was to demonstrate that MELK expression is upregulated in ER-negative breast cancers, and to test the hypothesis that MELK is a critical regulator of ER-negative breast cancer cell growth.

Methods: RNA from breast cancer cell lines and 60 human breast tumors was isolated for Q-PCR analysis of MELK. To determine if MELK regulates cell proliferation, ER-positive and ER-negative breast cancer cell lines were transfected with siRNA targeting MELK. Cells were replated 36 hours after transfection in 96-well plates at a density of 2,000/well. Cell number was measured by the MTS assay every day for 6 days. To determine if MELK levels are independently prognostic, we performed uni- and multivariate Cox regression analysis, including all available biological characteristics of the tumor in the model.

Results: MELK mRNA levels were significantly higher (p<0.02) in ER-negative breast cancers (20.2 ± 7 fold) compared to ER-positive breast cancers (5.0 ± 2 fold). In 6 of 7 ER-negative cell lines, MELK knockdown suppressed growth (≥50% growth inhibition) compared to cells transfected with control siRNA. MELK knockdown had little to no effect on growth of the 3 ER-positive cell lines tested. Importantly, in a multivariate Cox proportional hazards model, high MELK expression was shown to be independently associated with a poor prognosis, [HR=1.19 95% CI: (1.05, 1.36) p=0.007]. Current studies are focused on understanding how MELK regulates ER-negative cell growth, using ER-negative cell lines stably expressing an inducible dominant negative MELK protein.

Conclusions: MELK is highly expressed in ER-negative breast cancer, essential for ER-negative breast cancer cell growth, and is independently prognostic. These findings suggest that MELK is a promising target for the treatment of ER-negative breast cancer.

Supported by a Susan G. Komen for the Cure Promise Grant. (KG081694).

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 1664. doi:10.1158/1538-7445.AM2011-1664
Abstract 1669: Expression of a novel zinc-finger transcription factor ZKSCAN3 in human tumors and its potential as a therapeutic target
Authors: Yang Lin , Orlowski Robert Z.
Published: June 2012

Previous studies from our group demonstrated that zinc finger with KRAB and SCAN domains 3 (ZKSCAN3), a novel zinc-finger transcription factor, coordinated a gene expression program targeting Cyclin D1, Cyclin D2, NF- κB, Integrin β4, as well as VEGF and IGF-2, among others, in which these genes have been suggested to play critical roles in tumor progression. To examine if ZKSCAN3 represents a potential cancer therapeutic target, expression of ZKSCAN3 was investigated in multiple tumor types. By immunohistochemistry (IHC), we observed that ZKSCAN3 protein greatly expressed in majority tumor tissues and cells, including solid tumors (colorectal carcinoma, prostate cancer, bladder cancer, and breast cancer) and liquid malignancies (multiple myeloma, non-Hodgkin B cell lymphoma, mantle cell lymphoma, MALT, diffuse large B cell lymphoma), but not non-malignant adjacent tissues. ZKSCAN3 expression was also associated with tumor progression, evidenced by IHC staining in different stages of colorectal carcinoma and prostate cancer samples. Very interestingly, lack of ZKSCAN3 expression was observed in Hodgkin lymphoma and angioimmunoblastic T cell lymphoma, suggesting that ZKSCAN3 selectively expressed in certain tumor types. Moreover, quantitative real-time polymerase chain reaction (qPCR) was performed to verify ZKSCAN3 mRNA expression in multiple myeloma. Consistent with the IHC data, CD19+ normal B cells and CD138+ plasma cells have little expression of ZKSCAN3, meanwhile malignant plasma cells greatly expressed ZKSCAN3 gene. The differential expression pattern of ZKSCAN3 between tumor cells and non-malignant cells strongly suggests that ZKSCAN3 may play an important role in tumor pathogenesis. Indeed, in addition to the identified gene expression program that was regulated by ZKSCAN3, targeting ZKSCAN3 by its small interfering (si) RNA led to slowed tumor growth and decreased chemosensitivity, and overexpression of ZKSCAN3 greatly promoted tumor growth and enhanced chemosensitivity in the models of multiple myeloma and colon cancer, indicating that ZKSCAN3 may represent a promising cancer therapeutic target.

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 1669. doi:10.1158/1538-7445.AM2011-1669
Abstract 1671: GSK3-beta as a therapeutic target in bladder cancer
Authors: Becker Marie N. , von Roemeling Christina A. , Copland John A. , Williams Christopher R.
Published: June 2012

Bladder cancer is the 5th most commonly diagnosed cancer in the United States. Because superficial bladder cancer (SBC) recurs in about 60% of cases, standard treatment involves tumor resection followed by intravesical therapies to prevent recurrence, as well as lifelong periodic cystoscopy. There is an urgent need for better intravesical therapies to prevent both the recurrence and progression of SBC. Recent evidence has demonstrated that glycogen synthase kinase – 3 beta (GSK3β) plays an important role in pancreatic, ovarian and colon cancers; however, to date the possible role of this important kinase has not been examined in bladder cancer. We propose that GSK3β plays a cancer promoting role in bladder cancer and that inhibition of GSK3β will reduce bladder cancer growth. The GSK3β inhibitor, AR-A014418, has been demonstrated to reduce tumor growth in the previously cited cancers. AR-A014418 reduced cell proliferation of three bladder cancer cell lines (HT1376, T24 and UM-UC-3) with EC50 values of 17.5μM for HT1376, 8μM for T24, and 10μM for UM-UC-3 cells. The effect of AR-A014418 on downstream targets in these cell lines was evaluated by Western blots which indicated an increase in apoptosis in these cells in response to drug treatment as demonstrated by the presence by both PARP and caspase 3 cleavage. The NFkB downstream target XIAP also exhibited reduced protein levels after drug treatment. The HT1376 cell line was transduced with luciferase containing lentivirus particles to generate a luciferase expressing cell line, HT1376-LUC to use for both ectopic and orthotopic models of bladder cancer growth. The efficacy of AR-A014418 in reducing bladder cancer tumor growth was tested in an ectopic mouse model. HT1376-LUC cells mixed with matrigel were transplanted into the right flank of 50 female nude mice. After one week, mice containing tumors with an average volume of 52 mm3 were randomized into four treatment groups of 10 mice each. Mice were treated twice weekly via IP injection with placebo, 1, 5 or 7mg/kg AR-A014418. Tumor sizes were measured and imaged for luciferase activity once weekly. Tumor growth was inhibited by both 5mg/kg and 7mg/kg doses by 19 days of treatment (p < 0.05, Student's T test). Together, the in vitro and in vivo data suggest that GSK3β is a novel target for bladder cancer treatment. Future studies using an orthotopic model of bladder cancer are planned.

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 1671. doi:10.1158/1538-7445.AM2011-1671
Abstract 1672: Mitochondrial and bacterial peptides act on the formyl peptide receptor (FPR) to promote migration and proliferation in high grade glioblastoma cells
Authors: Boer Jennifer C. , Domanska Urszula M. , Kruizinga Roeliene C. , Boer Ingrid , de Haas Carla J.C. , de Vries Elisabeth G. , Den Dunnen Wilfred F.A. , van Strijp Jos A.G. , Walenkamp Annemiek M.
Published: June 2012

Background: Recently several aberrant molecular pathways and underlying genetic defects are elucidated in astrocytomas, among which the role of the formylpeptide receptor (FPR) malignant behavior. In a highly malignant human astrocytoma cell line FPR promotes cell motility, growth, and angiogenesis by interacting with host-derived agonists originating from necrotic cell material. Until now the only known ligands of FPR are N-formyl peptides, which are cleavage products of bacterial proteins like N-formyl-methionyl-leucyl-phenylalanine (fMLF). In humans the only conserved N-formyl peptides are the cleavage products of mitochondrial proteins. This study aims to identify these peptides as responsible ligands for FPR expressing high grade glioma cells.

Methods: We used FITC-fMLF and flow cytometry to confirm binding to FPR-expressing high grade human gliobastoma cell line U87. In addition, U937 cells (human promonocytic cell line) transfected with the FPR (U937FPR) and empty expression vectors were generated. The hallmark of chemokine receptor activation is a rapid and transient increase in the free intracellular Ca2+ level upon ligand binding. Formylated mitochondrial peptides fMLKLIV/fMLALV and FPR's natural peptide ligand fMLF were used to induce Ca2+ mobilization, chemotaxis as determined by the Neuro Probe® system and proliferation in U87 cells, analyzed by MTT assay.

Results: FITC-fMLF bound to FPR expressing U87 and U937FPR cells. Increasing concentrations of fMLF (10−8 M-10−5 M) caused a dose dependent calcium mobilization in FPR expressing U87 cells. Increasing concentrations of fMLF/fMLKLIV/fMLALV (10−8 M- 10−5M) were tested in U937FPR cells, again showing dose dependent calcium mobilization. In the chemotaxis assay, respectively 22, 20 and 24% of U937FPR cells migrated towards fMLF/fMLKLIV/fMLALV as compared to 4% towards 0.05% FCS. A 2.5 fold increase in migration of U87 cells towards 10−7 M fMLF was found as compared to 0.05% FCS. Respectively 20, 21 and 15% increase in U87 cell proliferation was found when cells were stimulated with 10−6 M fMLF/fMLKLIV/fMLALV.

Conclusions: These results indicate that mitochondrial peptides present in necrotic tumor material serve as ligands for FPR, expressed on high grade human gliobastoma cell line U87. Activation of FPR via N-formyl peptides causes increased cell growth and motility and is a potential target for anti-cancer therapy.

Supported by grant RUG 4622 of the Dutch Cancer Society

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 1672. doi:10.1158/1538-7445.AM2011-1672
Abstract 1678: Visualization of thymidine kinase 1 on the surface of cancer cell lines: A potential diagnostic marker and therapeutic target
Authors: Whitehurst Robert A. , Estevez Dagoberto , Sharp Daniel W. , Alegre Melissa M. , Robison Richard A. , O'Neill Kim L.
Published: June 2012

Thymidine Kinase 1(TK1) is a salvage pathway enzyme that has been shown to be elevated in many different cancer types, including breast, prostate, and colon. Previous research in our lab using plasma membrane separation has shown an increase in TK1 levels associated with the plasma membrane of cancer cells when compared to normal lymphocytes. After membrane separation the plasma membrane of normal lymphocytes showed a TK1 activity of 1,012 CPM/mg protein/hr(N=20) whereas those of cancer cells showed: Jurkat 34,096(N=28), Raji 70,195(N=20), HL60 46,217(N=34), and MDA-MB-435 50,359(N=24). In this study we sought to further investigate this association by visualizing the surface staining of fluorescently tagged antibodies to TK1. Using a primary rabbit anti-TK1 antibody and a secondary goat anti-rabbit antibody conjugated with a FITC fluorochrome we were able to stain the cells and identify TK1 bound to the cell surface. Using a fluorescent microscope we were able to visualize the fluorescent staining of TK1 in various cancer cell lines, including: Raji, Jurkat, HL60, MDA-MB-435, H358, and MCF-7. We compared these results to our control of stained normal lymphocytes, which showed significantly greater levels of staining in cancer cells. Our results further confirm the presence of membrane associated TK1, and provide further evidence of TK1's usefulness as a diagnostic marker and potential therapeutic target.

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 1678. doi:10.1158/1538-7445.AM2011-1678
Abstract 1682: Identification of RAM (ribonucleoprotein associated molecule) overexpressed in high-grade prostate cancer cells
Authors: Anchi Takashi , Tamura Kenji , Furihata Mutsuo , Satake Hirofumi , Ashida Shingo , Yamasaki Ichiro , Inoue Keiji , Shuin Taro
Published: June 2012

One of the most important problems in hormonal therapy for prostate cancer is the acquirement of hormone-resistant and/or androgen-independent phenotypes. These cells are high-grade and lead to the eventual death. To characterize the molecular features of high-grade prostate cancer, we performed a gene-expression profile analysis by cDNA microarray coupled with laser microbeam microdissection from 10 frozen high-grade prostate cancer tissues and found dozens of trans-activated genes. Among of them, we identified RAM as an overexpressed gene in high-grade prostate cancers. The function of this protein is unknown, but it has a domain structure relating to mRNA splicing. We demonstrated that RAM protein was localized in the nucleus. Knock-down of RAM expression by siRNAs were observed a significant decrease of cell numbers. In contrast, RAM overexpression promoted cell proliferation. These findings suggest that RAM could be involved in cell survival and/or proliferation of prostate cancer, and that it should be a potential molecular target for development of novel therapeutics.

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 1682. doi:10.1158/1538-7445.AM2011-1682
Abstract 1683: High throughput membrane proteomics for the discovery of oncology targets ideally suited for the development of antibody drug conjugates (ADCs)
Authors: Terrett Jonathan A. , Bozhenok Ludmila , Belanger Blake , Montes Inigo , Aud Dee , Ackroyd Jim , Anderson Amanda , Trang Michael , Barnes Martin , Lamble Harry , Yu Xiaohong , Rohlff Christian
Published: June 2012

Antibody Drug Conjugates (ADCs) are showing tremendous promise for cancer patients as recently demonstrated by the dramatic responses reported in breast cancer and Hodgkin's Lymphoma clinical trials to the ADCs T-DM1 and SGN-35. We have developed proteomics methods to enrich for membrane proteins that are expressed preferentially in cancer cells over normal cells. Potential ADC targetable proteins are then validated using the proprietary OGAP database as well as conventional expression analysis tools. The proteomics processes have identified novel cancer specific proteins as well as cancer specific isoforms of known proteins. These targets are then combined with large panels of antibodies to discover potential therapeutics with properties desirable for the development of ADCs. Antibodies are selected which : a) preferentially bind to tumor cells over normal cells, b) have good specificity and affinity, c) show high prevalence in target cancer indications by IHC, d) internalize on antigen binding, e) bind to relevant tox species tissues as they bind to human tissues. Here we describe the proteomics processes and use of the OGAP database and demonstrate the development of candidate therapeutic antibodies with the potential to become ADCs, which are directed to novel targets and cancer specific isoforms.

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 1683. doi:10.1158/1538-7445.AM2011-1683
Abstract 1696: Regulation of sensitivity to the platinum-containing drugs by the sec61 translocon
Authors: Abada Paolo B. , Howell Stephen B.
Published: June 2012

The Sec61 protein translocon is a multimeric complex that allows the transport of proteins across or laterally into a lipid bilayer. Components of the sec61 complex are involved in retrotranslocation, Golgi structure and vesicle transport. We have discovered that one component of this complex, the sec61β subunit, modulates cellular sensitivity to the platinum-containing drugs. As part of an effort to enhance sensitivity to the platinum drugs we sought to determine the mechanism of this effect. Sec61β expression in human 2008 ovarian cancer cells was constitutively knocked down by infection with a lentivirus producing an shRNA targeting sec61β, and knockdown was confirmed by qRT-PCR and western blot analysis which demonstrated a 96% reduction in mRNA expression and 87% reduction at the protein level. Effects on drug sensitivity were measured using an SRB assay, and platinum drug accumulation was quantified by inductively coupled plasma mass spectrometry. Sec61β knockdown (KD) did not alter cell viability or growth rate but resulted in 8-, 16.8- and 9-fold resistance to cisplatin, carboplatin, and oxaliplatin, respectively. Resistance to several other clinically relevant chemotherapeutics including paclitaxel, doxorubicin, vincristine, and etoposide was also increased but to a smaller extent (1.7 to 7-fold). Sec61β KD reduced the cellular accumulation of cisplatin to 67% of that in the parental cells. Given that transporters and chaperones that manage copper homeostasis regulate platinum drug accumulation and efflux, their expression in 2008 sec61β KD cells was analyzed by western blot. Among these, ATP7A was found to be 2-fold over-expressed while no change in ATP7B, ATOX1, or CTR2 could be detected. This increase in ATP7A expression was not altered by pretreatment with copper, cisplatin, or the copper chelator bathocuproine disulfonate. We conclude that the sec61β translocon modulates the cytotoxicity of many chemotherapeutic agents with the largest effect being on sensitivity to the platinum drugs. Sec61β regulates expression of the copper export transporter ATP7A that we have previously shown to control platinum drug sequestration and efflux.

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 1696. doi:10.1158/1538-7445.AM2011-1696
Abstract 1704: A role for glucosylceramide synthase in resistance to oxaliplatin in colorectal cancer
Authors: Madigan James P. , Rosenberg Daniel W.
Published: June 2012

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the U.S. Nearly 25 percent of patients who present with CRC have already developed metastatic disease. While combination therapies for metastatic CRC have improved median survival times, most patients inevitably become resistant to chemotherapy. Chemoresistance is thus the major cause of treatment failure in CRC patients, and accordingly, the development of novel therapies aimed at overcoming chemoresistance is of paramount importance. Many commonly used chemotherapeutic agents act by inducing cell death by increasing cellular levels of the bioactive sphingolipid, ceramide. In addition to serving well-known structural functions in the cell membrane, sphingolipids are increasingly recognized as important cellular signaling molecules. Studies have demonstrated that human colorectal tumors contain decreased concentrations of ceramide in comparison to normal colonic mucosa. Furthermore, multi-drug resistant cancer cells often display an altered sphingolipid content, with the presence of increased levels of the pro-survival ceramide metabolites. In this study we have examined the role of the ceramide-metabolizing enzyme, glucosylceramide synthase (GCS), in the mechanism of resistance to oxaliplatin. These studies utilize a series of oxaliplatin-resistant isogenic colorectal cancer cell lines. While much evidence has been presented establishing the role of GCS in resistance to a variety of chemotherapeutic agents, a role for GCS in oxaliplatin resistance has yet to be examined. Our studies demonstrate that oxaliplatin-resistant CRC cell lines have ∼ 2-fold more GCS protein with increased levels of the pro-survival ceramide metabolite, glucosylceramide, in comparison to the parental cell lines. We further demonstrate that inhibition of GCS by either pharmacological inhibitors or an RNAi-mediated gene knockdown results in reduced cellular glucosylceramide levels, with a concomitant increased sensitivity towards oxaliplatin. Furthermore, inhibition of GCS causes a reversion of an observed epithelial-mesenchymal transition phenotype observed in oxaliplatin-resistant CRC cell lines. Our results may offer a possible strategy with which to augment chemotherapeutic drug effectiveness in CRC.

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 1704. doi:10.1158/1538-7445.AM2011-1704
Abstract 1705: Comparison of uptake mechanisms for different anthracyclines in leukemic cells
Authors: Karim Hazhar , Bogason Alex , Vitols Sigurd
Published: June 2012

Aims: The mechanisms behind cellular anthracycline uptake are not completely understood. It is of great interest to gain more insight into cellular uptake mechanisms since this could possibly be used to increase the selectivity of the drugs. We therefore compared the uptake of 5 different anthracyclines, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by leukemic cells and also investigated the possible involvement of specific carriers.

Methods: HL-60 cells were incubated with increasing concentrations of anthracyclines for 1 hour and cellular drug uptake was determined. The cells were also preincubated with various protein inhibitors or nucleosides and then with the anthracyclines after which drug uptake was determined with HPLC. Apoptosis was determined with propidium iodine staining and flow cytometry.

Results: Of all anthracyclines studied DNR, IDA and PIRA had the highest cellular uptake with a sharp increase in uptake at extracellular concentrations > 1 µM. Uptake of DOX, DNR and IDA was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and dipyridamole, a nucleoside transport inhibitor, only inhibited the uptake of DNR. The addition of nucleosides reduced the uptake of DNR, IDA and PIRA.

Conclusion: Our results suggest different uptake mechanisms for various anthracyclines. We found evidence for carrier mediated uptake mechanisms, the data support the involvement of the nucleoside transporter family. Furthermore, our results also indicate an involvement of purinergic-2-receptor signalling in anthracycline uptake.

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 1705. doi:10.1158/1538-7445.AM2011-1705
Abstract 1708: Induction of GDF15 by cisplatin triggers apoptotic cell death in ovarian cancer cells
Authors: Izaguirre Daisy I. , Zu Zhifei , Tsang-Lee Yvonne T. , Mok Samuel , Wong Kwong-Kwok
Published: June 2012

Ovarian cancer is the fifth most common cause of death among women. The overall five year survival is 45.6% as a result of late diagnosis when treatment is mostly ineffective. Current treatment consists of complete resection followed by a combination of a taxane and a platinum -based chemotherapy regardless of the subtype. After standard treatment, 70% of patients will develop chemoresistance, creating a major challenge in the treatment of this type of cancer. Therefore, there is great interest in understanding the mechanisms by which tumors develop resistance. From the microarray data of a high grade serous ovarian cancer cell line (A2780) treated with varying concentrations of cisplatin and at varying time points, we identified four genes (CDKN1A, FDXR, TP53I3 and GDF15) which were significantly up-regulated by cisplatin in a time and dose dependent manner. The time and dose dependent upregulation of these genes were further validated by real-time RT-PCR and Western blot analysis in A2780 and two other ovarian cancer cell lines. We further tested whether GDF15 expression correlated with cisplatin response. RNA and protein from 6 different cell lines with different IC50 to cisplatin revealed a positive correlation between GDF15 induction and sensitivity to cisplatin. To further investigate the role GDF15 plays in the chemoresponse of ovarian cancer cell, we knocked down the expression of GDF15 by shRNA. Ovarian cancer cells with GDF15 knockdown showed a decrease in PARP cleavage, suggesting a pro-apoptotic role for GDF15. Thus, our results suggest that induction of GDF15 by cisplatin is one of the mechanisms that triggers apoptotic cell death in ovarian cancer cells. On the other hand, we were not able to detect an induction of GDF15 by cisplatin in a cisplatin resistant cell line A2780cp20. Thus, the lost of GDF15 induction could be one of the mechanisms of developing chemoresistance. Future studies to decipher the mechanism by which cisplatin induces GDF15 expression have the potential of identifying new targets that could circumvent chemoresistance to cisplatin.

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 1708. doi:10.1158/1538-7445.AM2011-1708
Abstract 1710: Expression of erbB3 upregulates Survivin via transcription-independent mechanism in erbB2-overexpressing breast cancer cells to confer Paclitaxel resistance
Authors: Wang Shuiliang , Huang Xiaoping , Lee Choon-Kee , Liu Bolin
Published: June 2012

The erbB2 receptor is often activated via dimerization with another erbB receptor, such as EGFR or erbB3. Studies show that co-expression of erbB3 and erbB2 is frequently observed in breast cancer; and erbB2 requires erbB3 to stimulate breast cancer cell proliferation. Increased resistance to therapeutic agents such as paclitaxel is one of the mechanisms by which erbB2 contributes to breast tumorigenesis. While several molecular mechanisms contributing to paclitaxel resistance have been proposed, a series studies demonstrate that overexpression of erbB2 inhibits paclitaxel-induced apoptosis in breast cancer cells. However, the role of erbB3 in paclitaxel resistance remains unclear. Our current studies focus on determining the role(s) of erbB3 in erbB2-mediated paclitaxel resistance in breast cancer cells.

Overexpression of exogenous erbB3 via either stable or transient transfection in erbB2-overexpressing, but not EGFR-expressing, breast cancer cells significantly decreases paclitaxel-induced growth inhibition and apoptosis. Consistently, knockdown of erbB3 expression with a specific shRNA in breast cancer cells with coexpression of both erbB2 and erbB3 enhances paclitaxel-induced apoptosis evidenced by increased DNA fragmentation, PARP cleavage, and activation of caspase-3, and -8. Furthermore, while forced overexpression of erbB3 increases, specific knockdown of erbB3 decreases the protein but not mRNA expression levels of Survivin only in the erbB2-overexpressing breast cancer cells. Targeting Survivin with specific shRNA overcomes paclitaxel resistance without effect on the expression levels of either erbB2 or erbB3. Mechanistic studies indicate that the specific PI-3K, Akt, and mTOR inhibitors, but not the MEK inhibitor, not only abrogate erbB3-mediated upregulation of Survivin, but also reinforce the erbB2/erbB3-coexpressing breast cancer cells to paclitaxel-induced growth inhibition.

Our studies demonstrate that heterodimerization of erbB2/erbB3 results in paclitaxel resistance in breast cancer cells via PI-3K/Akt/mTOR signaling pathway. Elevated expression of erbB3 upregulates Survivin through transcription-independent mechanism in erbB2-overexpressing cancer cells, suggesting that novel strategies targeting erbB3 or Survivin protein translation may enhance the efficacy of chemotherapeutic agents against breast cancer with erbB2-overexpressing 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 1710. doi:10.1158/1538-7445.AM2011-1710
Abstract 1717: SATB1 expression increases resistance of bladder cancer cells to cisplatin
Authors: Choudhary Shilpa , Voznesensky Olga , Pilbeam Carol , Taylor John A.
Published: June 2012

Background: SATB1 (special AT-rich sequence binding protein-1) is a nuclear protein involved in chromatin organization and gene regulation. Increased expression of SATB1 in breast and hepatocellular cancer cells has been shown to promote metastasis and multidrug resistance. We explored the effects of SATB1 expression in the high grade human bladder cancer cell line, HTB-5.

Methods: HTB-5 cells were transfected with an shRNA construct for SATB1 or a non-silencing (NS) control. Nuclear protein and mRNA expression were measured by Western analysis and real-time PCR, respectively. Cells were counted on days 2-6 of culture, and DNA replication was measured by thymidine incorporation (TdR) on day 3. Cell cycle distribution and cell viability were analyzed by propidium iodide-Annexin V FITC flow cytometry in 3 independent experiments. Cells were treated with cisplatin (6 μM) for 3 days.

Results: SATB1 mRNA and protein expression levels were 28-fold and 5-fold higher, respectively, in HTB-5 cells compared to UROtsa cells, a benign transformed human urothelial cell line. Expression of SATB1 was reduced 80% or more in shRNA transfected HTB-5 cells. Cell counts and TdR were decreased 20-23% (p<0.01) in SATB1 knockdown cells compared to NS controls. SATB1 knockdown was associated with a significant increase of 15% in G1/G0 and decrease of 18% in S-phase. There was no significant difference in cell viability between SATB1 knockdown cells and NS controls. However, cisplatin treatment resulted in a decrease in live cells from 61.9 ± 4.6% to 39.5 ± 5% (p<0.01) and an increase in apoptotic cells from 33.1 ± 4.4% to 56.1 ± 4.0% (p<0.01) in SATB1 knockdown cells compared to NS controls.

Conclusions: SATB1 expression was higher in a malignant compared to benign urothelial cell line and was associated with increased proliferation and increased resistance to chemotherapy.

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 1717. doi:10.1158/1538-7445.AM2011-1717
Abstract 1718: Exploring temozolomide resistance in glioblastoma using a combinatorial zinc-finger library approach
Authors: Narayanan Radhakrishnan , Levin Benjamin S. , Maeder Morgan L. , Joung J Keith , Nutt Catherine L. , Louis David N.
Published: June 2012

Recent therapeutic advances have improved control of glioblastomas, with a significant fraction of tumors responding to initial therapies. Unfortunately, all glioblastomas recur and lead to patient death. In an effort to better understand therapeutic resistance, we have explored high-dimensional profiling studies to understand mechanisms of therapeutic resistance and to identify novel therapeutic targets. Temozolomide (TMZ) is an alkylating chemotherapeutic agent that is used to treat all glioblastomas. Recurrent glioblastomas, however, become resistant to the effects of TMZ through a mechanism associated with inactivation of MSH6, a member of the mismatch repair gene family; MSH6-deficient glioblastomas grow more rapidly during TMZ therapy. To explore pathways of therapeutic resistance, we have designed an unbiased drug resistance selection screen to create TMZ-resistant human glioblastoma cell lines using combinatorial zinc-finger transcription factors (ZTFs). The combinatorial ZTF library enables activation of a gene or a set of genes that will be selected for resistance to TMZ. We have isolated resistant clones in the TMZ-sensitive A172 and Gli36 cell lines and present molecular profile analysis of these clones. By comparative gene expression profile analysis of novel ZTF-induced TMZ resistant glioblastoma cell lines, we expect to identify molecular pathways that confer TMZ resistance. In doing so, we also aim to identify possible biomarkers for TMZ resistance in recurrent glioblastomas.

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 1718. doi:10.1158/1538-7445.AM2011-1718
Abstract 1720: Tyrosine phosphorylation of HSC70 located in the cell membrane may regulate methotrexate transportation in murine L1210 leukemia cells
Authors: Liu Tuoen , Rios Zech , Sheridan Peter P. , Bhushan Alok , Lai James C.K. , Daniels Christopher
Published: June 2012

Bhushan and co-workers previously described an alteration in an unidentified 66-68 kDa membrane-associated, tyrosine-phosphorylated, methotrexate (MTX)-binding protein in the L1210 murine leukemia cell line. This protein was absent, at least in the tyrosine phosphorylated form, in cisplatin-resistant, methotrexate cross-resistant L1210/DDP cells. The 66-68 kDa protein was isolated by affinity chromatography, first on MTX-agarose followed by purification using an anti-phosphotyrosine immunoaffinity column. Amino acid sequence analysis identified the protein as heat shock cognate protein 70 encoded by the HSPa8 gene. The HSPa8 gene was found to be transcribed in both sensitive and resistant cell lines by RT-PCR and the sequence of the HSPa8 gene was identical in both cell lines. Western blot analysis showed that HSC70 was also expressed in both cell lines. A binding pull-down assay with MTX-agarose beads, followed by western blotting for HSC70, showed HSC70 to be present in cell lysates and in the MTX-binding fraction. Fractionation of cell homogenates into membrane and cytosolic fractions showed that HSC70 was found in both fractions but was not detected in the MTX-binding assay with the membrane fraction from the L1210/DDP cells, suggesting HSC70 located in the membrane fraction may be controlling MTX transport in the sensitive cells. Comparing the tyrosine phosphorylation status between L1210/0 and L1210/DDP cells, a band around 70 kDa was observed in the membrane fraction of L1210/0 cell but was not present in the L1210/DDP cells. Additionally, immunoprecipitation with anti-phosphtyrosine (PY20) agarose beads and detection of HSC70 by western blot showed little to no PY-HSC70 in the membrane fraction from the L1210/0 cells. Thse results suggest that HSC70 is a putative MTX binding protein, and HSC70 associated with the cell membrane may regulate methotrexate uptake. Tyrosine phosphorylation may contribute to the function of HSC70 in the cross-resistance mechanism in the murine L1210 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 1720. doi:10.1158/1538-7445.AM2011-1720
Abstract 1726: Overcoming multi-drug resistance with Paclitaxel-loaded polymer nanoparticles
Authors: Lee Yeonju , Graeser Ralph , Kratz Felix , Geckeler Kurt Ernst
Published: June 2012

Multi-drug resistance against chemotherapeutic agents is a major problem in the treatment of cancer. It is primarily mediated by over-expression of P-glycoprotein (P-gp) encoded by the MDR-1 gene by the tumor cells. Most of the promising anticancer drugs such as doxorubicin, docetaxel, paclitaxel, and mitoxantrone are P-gp substrates. Thus, various ways for overcoming the multi-drug resistance, e.g. combination of an anticancer drug and a P-gp inhibitor or the formulation of nanoparticles in conjunction with anticancer drugs as well as the P-gp inhibitor, have been studied. Anticancer drug-loaded nanoparticles, such as paclitaxel-loaded polymer nanoparticles, are excellent candidates to overcome multi-drug resistance due to their enhanced therapeutic efficacy induced by increased cellular accumulation and sustained intracellular drug delivery.

In this work, we developed an ampholytic copolymer that can encapsulate paclitaxel, and the resulting nanoparticles were found to have good water-solubility. In addition, in contrast to native paclitaxel, the paclitaxel-loaded nanoparticles were able to reverse multi-drug resistance in MCF7/ADR and MT-3/ADR cell lines, resulting in drug sensitivities comparable to the parental MCF7 and MT3 cell lines. P-gp expression was confirmed in both ADR cell lines by Western blotting, and its functionality was verified using a rhodamine accumulation and efflux assay. The nanoparticles were taken up by endocytosis in both cell lines and their enhanced therapeutic efficacy is probably due to the different mode of entry into the cell that may avoid the P-gp efflux pump. As a consequence higher concentrations of paclitaxel reach the nucleus and induce cell killing.

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 1726. doi:10.1158/1538-7445.AM2011-1726
Abstract 1730: Transient PI3K inhibition induces apoptosis and overcomes HGF-mediated resistance to EGFR-TKIs in EGFR mutant lung cancer
Authors: Yano Seiji , Donev Ivan S. , Wang Wei , Li Qi , Takeuchi Shinji , Yamada Tadaaki
Published: June 2012

Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, show favorable response to EGFR mutant lung cancer. However, the responders acquire resistance almost without exception. We recently reported that hepatocyte growth factor (HGF) induces EGFR-TKI resistance by activating MET which restores downstream MAPK-ERK1/2 and PI3K-Akt signaling. The purpose of this study was to determine whether inhibition of PI3K, a downstream molecule of both EGFR and MET, could overcome HGF-mediated EGFR-TKI resistance in EGFR mutant lung cancer cells, PC-9 and HCC827. Experimental Design: We explored therapeutic effect of a class I PI3K inhibitor PI-103 on HGF-induced EGFR-TKI resistance in vitro and in vivo. Results: Unlike gefitinib or erlotinib, continuous exposure with PI-103 inhibited proliferation of PC-9 and HCC827 cells, even in the presence of HGF. On the other hand, in gefitinib-resistant xenograft model using PC-9 cells mixed with HGF high producing fibroblasts, PI-103 monotherapy did not inhibit tumor growth. However, PI-103 combined with gefitinib successfully regressed gefitinib-resistant tumor. In vitro experiments considering short half life of PI-103 reveal that transient exposure of PI-103 combined with gefitinib caused sustained inhibition of Akt phosphorylation, but not ERK1/2 phosphorylation, resulting in induction of tumor cell apoptosis even in the presence of HGF. Conclusions: These results indicate that transient blockade of PI3K-Akt pathway by PI-103 and gefitinib could overcome HGF-mediated resistance to EGFR-TKIs by inducing apoptosis, further suggesting usefulness of double blockade of PI3K-Akt signaling at upstream (EGFR) and downstream (PI3K) for more successful treatment of EGFR mutant lung 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 1730. doi:10.1158/1538-7445.AM2011-1730
Abstract 1731: TAK-701, a humanized monoclonal antibody to HGF, reverses gefitinib resistance induced by tumor-derived HGF in non-small cell lung cancer with an EGFR mutation
Authors: Okamoto Wataru , Okamoto Isamu , Tanaka Kaoru , Arao Tokuzo , Nishio Kazuto , Fukuoka Masahiro , Jänne Pasi , Nakagawa Kazuhiko
Published: June 2012

Most non-small cell lung cancer (NSCLC) tumors with an activating mutation of the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib but ultimately develop resistance to these drugs. Hepatocyte growth factor (HGF) induces EGFR-TKI resistance in NSCLC cells with such a mutation. We investigated strategies to overcome gefitinib resistance induced by HGF. Human NSCLC cells with an activating EGFR mutation (HCC827 cells) were engineered to stably express HGF (HCC827-HGF cells). HCC827-HGF cells secreted large amounts of HGF and exhibited resistance to gefitinib in vitro to an extent similar to that of HCC827 GR cells, in which the gene for the HGF receptor MET is amplified. A MET-TKI reversed gefitinib resistance in HCC827-HGF cells as well as in HCC827 GR cells, suggesting that MET activation induces gefitinib resistance in both cell lines. TAK-701, a humanized monoclonal antibody to HGF, in combination with gefitinib inhibited the phosphorylation of MET, EGFR, ERK, and AKT in HCC827-HGF cells, resulting in suppression of cell growth and indicating that autocrine HGF-MET signaling contributes to gefitinib resistance in these cells. Combination therapy with TAK-701 and gefitinib also markedly inhibited the growth of HCC827-HGF tumors in vivo. The addition of TAK-701 to gefitinib is a promising strategy to overcome EGFR-TKI resistance induced by HGF in NSCLC with an activating EGFR mutation.

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 1731. doi:10.1158/1538-7445.AM2011-1731
Abstract 1786: Improved survival with gemcitabine and CTLA-4 blockade in a murine model of prostate cancer
Authors: Ariyan Charlotte E. , Quezada Segio , Corse Emily , Montalvo Welby , Allison James
Published: June 2012

Introduction: Immunotherapy with CTLA-4 blockade has resulted in dramatic responses in patients with advanced melanoma and prostate cancer. However, only 10-30% of patients have a response, therefore there is a need to increase the efficacy of CTLA-4 blockade. In this work we tested the hypothesis that combinatorial approaches incorporating tumor destruction with conventional chemotherapy and CTLA-4-blockade would synergize to significantly increase anti-tumor activity in vivo.

Experimental Procedure: Mice with a transplantable prostate tumor (TRAMPC2) were treated with 5 doses of gemcitabine alone, or followed by immunotherapy with CTLA-4 blockade. Control mice were treated with isotype antibody. Tumor growth was measured by calipers and mice were sacrificed when tumors reached 15mm. Progression was followed over time and CD8 T cell responses to the tumor specific antigen SPAS were examined by tetramer staining and functional assays ex vivo.

Data: Mice treated with the combination gemcitabine and α-CTLA-4 had improved survival over mice treated with gemcitabine alone. The majority of mice treated with control Hamster Ig or CTLA-4 blockade died (median survival 59 days for both groups). The median survival of mice treated with gemcitabine was 72 days with 50% having long-term survival. The addition of α-CTLA-4 to gemcitabine treatment increased median survival to > 125 days with 65% of mice with long term survival (p<0.05 for survival of gemcitabine versus gemcitabine + α-CTLA-4). The additive effect of α-CTLA-4 was lost when CD8 T cells were depleted. Analysis of draining lymph nodes revealed a reduced number of T regulatory T cells and myeloid suppressor cells with gemcitabine treatment. Mice receiving combination treatment had increased accumulation of tetramer positive CD8 cells for the SPAS peptide in the spleen and tumor draining lymph node. In addition, the combination treated mice had increased IFNγ production by ELISPOT in response to SPAS peptide re-stimulation ex vivo (p<0.05 for gemcitabine versus gemcitabine and α-CTLA-4).

Conclusion: Combination chemo- and immunotherapy with gemcitabine and α-CTLA-4 improves survival. This is associated with an accumulation of CD8 cells that are tumor specific. Depletion of CD8 cells reduces the efficacy of this treatment. This study supports further investigation into the additive effects of chemotherapy with CTLA-4 blockade.

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 1786. doi:10.1158/1538-7445.AM2011-1786