bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B3: Angiotensin IV-Related Hepatocyte Growth Factor Antagonists act as Anti-Angiogenic Anti-Cancer Agents
Authors: Kawas Leen H. , Ward Brandi C. , Mojica Chromewell , Wright John W. , Harding Joseph W.
Published: March 2013

Hepatocyte growth factor (HGF) promotes tumor angiogenesis by both vascular endothelial growth factor (VEGF)-dependent and VEGF-independent mechanisms. The VEGF-dependent action of HGF relies on its ability to induce VEGF expression while the VEGF-independent mechanism appears to involve the direct activation of Akt and ERK. This later mechanism may also be an important contributor to development of tumor resistance to anti-VGEF (anti-angiogenic) drugs. As such, the rational design of newer therapeutics that target and inhibit HGF may provide a more complete and long-lasting therapeutic effect. Angiogenesis, the development of new blood vessels, is essential for tumorigenesis. Tumor cells often up-regulate the production of pro-angiogenic growth factors such as the HGF. The angiotensin IV analog Norleual has been shown recently to act as an HGF/Met antagonist, inhibiting HGF dimerization and activation, blocking the binding of HGF to its Met receptor, impeding HGF-dependent activation of Met, and attenuating HGF-dependent cellular activities. Here we show for the first time that Norleual can inhibit the proliferation and migration of human endothelial cells. Further Norleual treatment results in a dramatic loss of deposited extracellular matrix proteins from both human endothelial and murine breast cancer cells. As anticipated from the abovementioned studies, Norleual markedly attenuated both ex vivo and in vivo angiogenesis. These results were extended to a murine breast cancer model where Norleual dramatically suppressed both tumor growth and vascularization. The major implication of this study is that molecules targeting the HGF/Met system like Norleual are powerful antiangiogenics and may be useful in combination with anti-VGEF drugs (multitargeted approach) to inhibit pathophysiological angiogenesis. Rational combination therapies may help in decreasing the possibility for drug resistance development and resolving issues related to toxicity.
Abstract B31: HuR cytoplasmic translocation and doxorubicin: how phosphorylation is involved in chemoresistance
Authors: Latorre Elisa , Quattrone Alessandro , Provenzani Alessandro
Published: March 2013

Introduction: HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we identified HuR as a key protein in the mechanisms leading to the insurgence of chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7) and investigated the signaling pathways responsible of its activation.

Material and methods: We challenged a small library of about 90 chemical compounds with a high-content screening assay to quantitavely measure HuR translocation. Hits pointed to PKCδ, Rho kinase and ERK as potential HuR regulators.

Results and discussion: We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. We identified HA1004, AG494, U0126, AG490 and Erbstatin compounds that could inhibit HuR cytoplasmic accumulation directly hampering HuR phosphorylation or indirectly blocking kinase pathways. Among the hits rottlerin showed to be the most effective in blocking HuR nuclear export and in having correspondingly antagonistic effects with doxorubicin on cell toxicity. Co-immunoprecipitation of PKCδ and HuR upon doxorubicin confirmed the validity of HCS indications. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets as TOP2A and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response.

Conclusions: The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin, that restoration of HuR expression in resistant cells resensitizes them to the action of this drug. Moreover we suggest a novel mechanism of pharmacoresistance based on the interplay among the doxorubicin target TOP2A, its post-transcriptionally regulator HuR and the signaling control of PKCδ.
Abstract B34: Bortezomib induces G2-M arrest through ROS-inducible phosphorylation of ATM-CHK1 in human colon cancer cells
Authors: Seung-Woo Hong , Won Kim Tae , Jai-Hee Moon , Yong-Sang Hong , Dong-Hoon Jin , Jae-Sik Shin , Hyun Yoon Dok , Kyu-pyo Kim , Jae-Lyun Lee , Jung Shin Lee
Published: March 2013

Colorectal cancer (CRC) is one of the most common cancer types, however, newer drug development has reached a plateau at present. Bortezomib (PS-341, Velcade®) is a proteasome inhibitor and approved in the treatment of hematologic malignancies including multiple myeloma. A few trials of bortezomib alone or combination chemotherapy for CRC patients have been reported, however, the results were not so satisfactory, and it might be due to the lack of understanding on the action mechanism. In this report, we suggest the possibility of using bortezomib as a proteasome inhibitor in human colon cancer cells. Although bortezomib has been known to induce apoptosis and cell cycle arrest in various human cancer cells, the inhibitory mechanism of it was not clear. First, bortezomib induced G2-M arrest. Its treatment causes an increase of intracellular reactive oxygen species (ROS) levels and activation of ATM-CHK1 pathway, but not ATR. Also, activation of that pathway led to inactivation of cdc2. Moreover, treatment with NAC, ROS scavenger, inhibited phosphorylation of ATM and subsequent to decrease of cell number at G2-M phase in response to bortezomib indicating that the increased level of ROS after exposure to bortezomib is a result of ATM phosphorylation. In addition, knockdown of endogenous ATM by RNA interference induced the decreased sensitivity to bortezomib. Therefore, these results suggest that bortezomib induces G2-M arrest through ROS-inducible ATM phosphorylation and shows antitumor activity in human colon cancer cells, implying a potential possibility of further investigations in the treatment of CRC patients.

Acknowledgement: This study was supported by grants from the Korea Health 21 R&D Project, Ministry of Health and Welfare and Family Affairs, Korea (A062254).
Abstract B35: Alternate receptor tyrosine kinases provide protection from FGFR inhibition in HNSCC cells as identified by genome-wide shRNA screening
Authors: Singleton Katherine R. , Tan Aik-Choon , Kim Jihye , Heasley Lynn
Published: March 2013

The efficacy of many biologically targeted cancer therapies is limited by intrinsic and acquired resistance. Head and neck squamous carcinoma (HNSCC) patients show only a 4–10% response to the epidermal growth factor receptor (EGFR) inhibitors, gefitinib and erlotinib, despite EGFR over-expression in over 90% of tumors. Our lab has previously shown that gefitinib insensitive non-small cell lung cancer (NSCLC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. RNAi-based loss-of-function screens are an important tool to identify synthetic lethal interactions between the inhibition of two genes in order to efficiently identify functional survival mechanisms and potent drug combinations. We deployed a whole genome screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ12908010, in HNSCC cell lines. We found that multiple alternate receptors provided protection from FGFR inhibition in these cell lines, including the receptor tyrosine kinases (RTKs) epidermal growth factor receptor 2 (ErbB2) and hepatocyte growth factor receptor (Met). We validated the results of the screen by showing that specific knockdown of either ErbB2 or Met in combination with FGFR inhibitor treatment led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ErbB2 or Met. The combination therapies did not lead to greater inhibition of growth in the cell lines where ErbB2 and Met were not identified as synthetic lethal with FGFR. We extended the study to find that the combination of knockdown of either ErbB2 or Met with FGFR inhibition decreased the growth of additional cell lines, indicating that the combination therapies could be broadly applicable. These results reveal a role for alternate RTKs in maintaining pro-growth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR and either ErbB family members or Met.
Abstract B40: Paracrine interactions through CXCL1/2 chemokines promote breast cancer metastasis and chemoresistance
Authors: Acharyya Swarnali , Oskarsson Thordur , Vanharanta Sakari , Malladi Srinivas , Kim Juliet , Brogi Edi , Norton Larry , Massague Joan
Published: March 2013

Cancer progression is a complex process that involves tumor growth at the primary site coupled with metastatic colonization and outgrowth at a distant organ site. In the case of solid tumors, 90% of deaths are known to occur from metastasis. Searching for mediators of breast cancer progression and metastasis, we identified a signature of 18 genes, the expression of which in primary tumors could predict the risk of lung metastasis recurrence in over 700 breast cancer patients. The sole chemokine in the lung metastasis signature is CXCL1, a member of the C-X-C family of chemokines. We find that increased expression of CXC family members could be explained in part by amplification of 4q21, a locus that harbors CXCL1-8 in breast tumors and metastases that we examined. Investigating the functional role of the C-X-C family in breast cancer metastasis led us to uncover a series of paracrine interactions mediated by CXCL1 and CXCL2 with components of the tumor microenvironment. Using breast cancer cell lines, primary patient derived cells and transgenic mouse models of mammary cancer and lung metastasis, we show that CXCL1/2 serve dual functions promoting both primary tumor growth and lung metastasis. We also demonstrate that CXCL1/2 recruits myeloid subpopulations that are essential in mediating cancer cell survival. Using small molecule inhibitors targeting CXCL1/2 signaling, we elucidate that this chemokine axis is critical in mediating breast cancer metastasis to lung and can be pharmacologically targeted.

The standard treatment for metastatic breast cancer is a combination of chemotherapeutic drugs. Disappointingly, these therapies only provide limited benefit and almost always result in relapse with acquired resistance and deadly consequences. We found that CXCL1, CXCL2 and other related family members were frequently upregulated in breast cancer models exposed to a variety of cytotoxic agents such as doxorubicin and taxanes. Moreover, our study revealed previously unknown tumor-stroma interactions that are initiated by CXCL1/2 in response to chemotherapy. Additionally, our functional studies in animal models and clinical evidence from patient samples demonstrated a function of CXCL1/2 in mediating chemoresistance. Interestingly, pharmacological inhibition of the CXCL1/2 paracrine axis in combination of chemotherapy significantly sensitized breast tumors to the therapy. In particular, combined treatment dramatically reduced the outgrowth of cancer cells at the lung metastatic site. Together, our findings reveal a network of tumor-microenvironment interactions mediated by the CXC chemokine axis that promotes tumor growth and metastasis on one hand and chemoresistance on the other.
Abstract B41: Thymoquinone induces-cell cycle non-specific-cell death in cisplatin-resistant ovarian cancer cell through up-regulation of PTEN expression
Authors: Arafa El-Shaimaa A. , Wani Altaf A.
Published: March 2013

Ovarian carcinoma is one of the most prevalent and lethal gynecological malignancies, and is the fifth leading cause of cancer death among women in the United Stat. Like most malignancies, ovarian cancer is a heterogeneous disease characterized by multiple molecular alterations. These include loss of function of tumor suppressor genes, deregulation of Bcl-2 family proteins, inhibition of caspase activation, and activation of cellular survival signaling pathways, such as HER-2/neu and phosphatidylinositol-3 kinase PI3K/Akt. Over years the use of natural compounds as anticancer agents acquired great importance. Thymoquinone (TQ) is the main bioactive component of the volatile oil of the black seed (Nigella sativa, Ranunclulaceae family). TQ has been used as antioxidant, anti-inflammatory, and antineoplastic medicines for more than 2,000 years. Previous studies reported that TQ exhibited inhibitory effects on cell proliferation of many types of cancer cell lines, including breast adenocarcinoma, lung adenocarcinoma, colorectal cancer, etc. Here, we report that TQ greatly inhibited cell proliferation of cisplatin-resistant ovarian cancer cells (A2780/CP70) 24, 48 and 72 hours after treatment in a concentration-dependent manner. However, our flow cytometry data revealed that TQ is a cell cycle non-specific for CP70 cells. Our results also showed that TQ upregulated PTEN and p53 in CP70 cells while downregulated phospho-Akt in a dose-dependent manner. Current investigations are ongoing to fully understand the mechanism of action of TQ in cisplatin-resistant ovarian cancer cells.
Abstract B44: Combination treatments of SCLC with ABT-263 and Immunotoxins produces synergistic killing of ABT-263-resistant cells
Authors: Mattoo Abid R. , Fitzgerald David J.
Published: March 2013

Small cell lung cancer (SCLC) cell lines are variably sensitive to the BH-3 only peptide mimetic, ABT-263. To determine their sensitivity to toxin-based reagents, we incubated four representative SCLC lines with a model Pseudomonas exotoxin-based immunotoxin directed to the transferrin receptor. Remarkably in 4-of-4 lines, there was little evidence of immunotoxin-mediated cytotoxicity despite near complete inhibition of protein synthesis. However, when combinations of ABT-263 and immunotoxin were added to the ABT-263-resistant cell lines (H196 and H69AR), there was synergistic killing as evidenced by increased activation of caspase 3/7, annexin V staining and loss of cell integrity. This synergy was also noted when the closely related compound ABT-737 was combined with the same immunotoxin. To establish that the synergy seen in tissue culture could also be achieved in vivo, H69AR cells were grown as tumors in nude mice and shown to be susceptible to the killing action of an immunotoxin-ABT combination but not to either agent alone. When immunotoxin-ABT combinations were added to ABT-263-sensitive lines (H146 and H889), killing was primarily due to the action of ABT-263. Our data support combination approaches for treating ABT-263-resistant SCLC with ABT-263 and a second agent that provides synergistic killing action.
Abstract B46: Targeting tumor-propagating cells in medulloblastoma through inhibition of G2/M cell cycle regulators
Authors: Markant Shirley , Esparza Lourdes Adriana , Barton Kelly , Sun Jesse , Wechsler-Reya Robert
Published: March 2013

Medulloblastoma is the most common malignant brain tumor in children. While the current treatment strategy of surgery plus radiation has a 60–80% cure rate, the survivors suffer severe side effects, including growth delays and cognitive deficits. Therefore, more effective, less toxic treatments are needed. One approach to achieving this is targeting the cells within a tumor that are capable of re-populating the tumor, known as tumor-propagating cells (TPCs). These cells are thought to be responsible for tumor resistance to conventional therapies. In the patched mutant mouse model of medulloblastoma, TPCs are marked by expression of the cell surface carbohydrate antigen CD15. Our previous studies have suggested that CD15+ cells display increased expression of genes associated with the G2/M phases of the cell cycle. Cell cycle analysis suggests that CD15+ cells move quickly through the G1 and S phases of the cell cycle, but accumulate in the G2/M phases. This accumulation in G2/M may represent a vulnerability of CD15+ TPCs, and inhibition of G2/M regulators, such as Aurora kinases, may represent one approach for targeting TPCs and overcoming therapeutic resistance. Treatment of tumor cells from patched mutant mice or human Hedgehog-associated medulloblastoma with Aurora kinase inhibitors in vitro leads to inhibition of proliferation, arrest in G2/M and apoptosis. Furthermore, in vivo treatment of tumor-bearing mice with Aurora kinase inhibitors significantly delays tumor progression and prolongs survival. Our findings suggest that targeting Aurora kinases in TPCs may represent a new approach for the treatment of human medulloblastoma.
Abstract B47: Identification of a novel ALK inhibitor active against Crizotinib-allele resistant mutants
Authors: Nicolas Jean-Paul , Calvet Loreley , Vergne Fabrice , Leroy Vincent , Garcia-Echeverria Carlos , Valtingojer Iris , Dassencourt Laurent , Herve Francoise , Serres Emerson , Delarbre Laure , Wolfrom Meredith , Lennon Amanda , Hoffmann Dietmar , Deprets Stephanie
Published: March 2013

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Activating mutations in ALK are oncogenic and cause 10% to 15% of neuroblastoma cases. Similarly, activating ALK fusions have been detected in several cancers, including ALCL (NPM-ALK in 70% of patients) and NSCLC (EML4-ALK in 3% to 7% of patients).

Several ALK inhibitors are currently undergoing clinical development, among which crizotinib has recently received marketing approval from the U.S. FDA under the name Xalkori. This nonselective ALK inhibitor has provided clinical benefit to lung cancer patients, but its sustained efficacy seems to be impaired by acquired drug resistance and several publications have already described ALK secondary mutations identified in patients who progressed while on crizotinib therapy.

In this study, we have:

Identified EML4-ALK mutations able to confer resistance to crizotinib.

A screen using the Ba/F3 system found 24 residues in the ALK kinase domain where mutations can cause crizotinib resistance. Some of these mutations (L1152R, C1156Y, F1174L, G1202R, S1206F) have been identified in lung cancer patients progressing under crizotinib treatment

Evaluated the activity of novel in-house and reference ALK kinase inhibitors against Ba/F3 cell lines stably expressing wild-type EML4-ALK and selected crizotinib-allele resistant mutantsPerformed in vivo head to head studies with a representative in-house ALK inhibitor and crizotinib to compare their pharmacodynamic properties

This work resulted in the identification of an ALK kinase inhibitor with similar potency against wild-type EML4-ALK and crizotinib-allele resistant mutants and displaying significant target coverage in in vivo settings.
Abstract B48: Sensitization of Hepatocellular Carcinoma Cells to TRAIL by a Novel Aκt/NF-kappaB Signaling Inhibitor
Authors: Omar Hany A. , Arafa El-Shaimaa A. , Weng Jing-Ru
Published: March 2013

Tumor necrosis factor-related apoptosis ligand (TRAIL) represents a promising cancer therapeutic agent considering its selectivity in targeting malignant cells while sparing normal cells. However, hepatocellular carcinoma (HCC) cells are intrinsically resistant to TRAIL, in part, due to the compensatory activation of nuclear factor-kappaB (NF-κB). Previously, based on the scaffold of indole-3-carbinol, we developed a potent Akt/NF-κB signaling inhibitor, OSU-A9, that exhibits in vitro and in vivo efficacy in suppressing HCC cell growth.

In this study, we report the sensitizing effect of OSU-A9 on TRAIL-induced cell death in Hep3B and Huh7 HCC cells by antagonizing TRAIL-induced NF-κB activation. Even at 1 μM, OSU-A9 could restore the sensitivity of these cells to TRAIL-mediated antitumor effect to a level comparable to that of TRAIL-sensitive PC-3 cells. Exposure to OSU-A9 led to a dose-dependent reduction in the expression and nuclear localization of RelA/p65 by suppressing Akt activation and RelA expression, which was accompanied by parallel decreases in the expression of antiapoptotic Bcl-xL, Mcl-1, cIAP1, cIAP2, and survivin. In addition, OSU-A9 upregulated death receptor (DR)5 expression via a reactive oxygen species-dependent mechanism. Concertedly, these effects gave rise to increased capsase-3 and caspase-8 activities and PARP cleavage in TRAIL-treated cells, indicative of therapeutic synergy between OSU-A9 and TRAIL. Moreover, this synergistic effect was abolished by the ectopic expression of NF-κB/p65, supporting the role of NF-κB inhibition in OSU-A9-mediated sensitization of HCC cells to TRAIL. Taken together, the ability of OSU-A9 to accentuate TRAIL-induced apoptosis by inactivating NF-κB signaling might foster a promising therapeutic strategy for HCC.
Abstract B54: Role of inverted Alu repeats in triggering TMPRSS2-ERG fusions in prostate cancer following irradiation
Authors: Singh Sheetal , Cheung Jean P. , Ho Bay , Vaughan Andrew , Ghosh Paramita
Published: March 2013

Radical Prostatectomy and radiation therapy are the two most common treatments for primary prostate cancer (PCa). However, patients treated with radiotherapy are at greater risk for metastases compared to those treated with radical prostatectomy. Among other factors regulating biochemical or clinical recurrence is the formation of gene fusions. Ionizing radiation (IR) can trigger formation of TMPRSS2-ERG in PCa which may facilitate the development of more aggressive clones, especially in men with an intact androgen axis. Bioinformatics analysis has indicated that breakpoint regions of TMPRSS2 and ERG are rich in Alu elements, including inverted Alu repeat that are inherently unstable when in close proximity. Previous work in multiple cell lines showed that adjacent inverted Alu repeats, but not tandem or single Alu's, are recombinationally unstable subsequent to IR exposure. Based on these results, we hypothesize that regions containing inverted Alu repeats in the prostate genome are vulnerable to DNA strand breaks and rearrangements in hormone naïve PCa patients undergoing IR therapy.

As gene fusions require each gene to be broken prior to fusion, the effect of irradiation on ERG fragmentation was studied using ligation mediated-PCR. Data from two androgen-sensitive PCa cell lines, PC-346C and LNCaP, showed that after irradiation DNA breaks were introduced preferentially in proximity to an Alu inverted repeat, Alu-Y pair, in intron 3 of ERG. To determine if the enhanced fragmentation observed in this region is translated into rearrangements, FISH (fluorescence in-situ hybridization) probes were designed to bracket the area of interest in ERG. In addition, the same region has been analyzed by inverse-PCR to detect ERG rearrangements. Application of these techniques will enable a better understanding of those mechanisms driving the formation of fusion genes in the proximity of Alu repeats.
Abstract B58: The ATP-competitive mammalian target of rapamycin (mTOR) inhibitors suppress proliferation and induce apoptosis in the acquired rapamycin resistant breast cancer cells
Authors: Tzung-Ju Wu , Chi-Kwan Tsang , Yan-Jie Zhang , Steven Zheng X.F.
Published: March 2013

The mammalian target of rapamycin (mTOR) is the major regulator of cell growth, metabolism and survival. The anti-cancer effect of allosteric mTOR-inhibitors, rapamycin and its analogs (rapalogs), has been tested extensively in different types of cancer cells. However, there is only limited clinical efficacy observed by treatment of these traditional mTOR inhibitors, partly due to the occurring of acquired rapamycin resistance (ARR). Several new mTOR inhibitors have been developed which specifically target to the catalytic sites of mTOR by competing with ATP binding, and are suggested to be more effective in inhibiting mTOR signaling and cell growth. However, their effect and mechanisms in ARR cells are not clear yet. In the present study, we used an in vitro model of acquired rapamycin-resistant (ARR) breast cancer cells to evaluate the efficacy of two major classes of ATP-competitive mTOR inhibitors, PI3K/mTOR dual and mTORC1/mTORC2 inhibitors. We found that ATP-competitive mTOR inhibitors acutely suppress the proliferation and prominently inhibit PI3K/mTOR signaling of ARR breast cancer cells. In contrast to the cytostatic effect of rapamycin, the ATP-competitive mTOR inhibitors induced apoptosis in ARR breast cancer cells. Unexpectedly, one of PI3K/mTOR dual inhibitors we tested, PI-103, only transiently inhibits its proposed signaling targets and the anti-proliferation effects are possibly mediated through the off-target mechanism. Our results support the rationale to use ATP-competitive mTOR inhibitors to treat rapamycin-resistant breast cancer cells and highlight the putative off-target effect of the new generation of mTOR inhibitor on cancer cells.
Abstract B59: MET gene copy number related to radiation treatment response and prognosis in breast cancer
Authors: Veenstra Cynthia , Pérez-Tenorio Gizeh , Fornander Tommy , Nordenskjöld Bo , Stål Olle
Published: March 2013

The hepatocyte growth factor receptor (HGFR), also known as the Met receptor, promotes cell survival and proliferation by activating signaling pathways such as the PI3K/Akt and RAS/MAPK pathway. HGFR, coded by MET, has been shown to be abundant in several cancers and in some cases it interferes with treatment response. In vitro, HGFR has been shown to be upregulated and activated after radiotherapy, increasing the protection from apoptosis and thus increasing radiation resistance. In this study, it was aimed to determine the number of MET gene copies in breast tumors and correlate these findings with different clinicopathological parameters that were available in this material. Tumors of 172 patients with primary breast cancer were analyzed for MET using qPCR. Increased MET copy number (three or more copies) was found in 34% of the tumors. Five or more gene copies were found in 4.7% of the patients and indicated a significantly higher risk of developing distant metastasis. For the patients that received radiotherapy as the only postoperative treatment, the number of MET copies was associated with increased risk of distant metastasis. In terms of locoregional recurrence, it was found that increased MET copy number was associated with worse response to postoperative radiation therapy compared with chemotherapy. These results suggest that radiotherapy in combination with a Met inhibitor might be an option for patients with increased MET gene copy number to overcome radiation resistance.
Abstract B6: PEGPH20 depletion of pericellular hyaluronan sensitizes high hyaluronan-producing tumor cells in antibody-dependent cell-mediated cytotoxicity
Authors: Singha Netai C. , Jiang Ping , Zimmerman Susan , Connor Robert , Frost Gregory I. , Huang Zhongdong , Shepard H. Michael
Published: March 2013

Monoclonal antibodies (MAbs) have been developed successfully as part of the anticancer armamentarium. However, even in cases where the target of the antibody is stably and abundantly expressed, efficacy often falls below expectations. The tumor microenvironment (TME) has previously been shown to contribute to resistance to MAb therapy because matrix-associated high interstitial fluid pressure inhibits efficient drug penetration (1). Hyaluronan is an important component of the extracellular matrix (ECM) in about 25% of tumors, and 56% of breast cancers. This glycosaminoglycan is produced by malignant and stromal cells and acts to cross-link ECM proteins, contributing to the desmoplastic phenotype. Accumulation of HA has been associated with more aggressive malignancy (2).In this work, we show that >50% of p185HER2-overexpressing breast tumors have a high accumulation of HA (HA+3). We hypothesized that tumor cells which overexpress HA create a protective “turtle shell” when it cross-links other ECM proteins (e.g., versican), and this pericellular structure protects the HER2-overexpressing tumor cell from attack by antibody-mediated cellular cytotoxicity (ADCC). SKBR3 (overexpressing p185HER2) and MDA-MB-231 (expressing EGFR) breast tumor cells were transfected with hyaluronan synthase 2 (HAS2), creating SKBR3/HAS2 (HA+3) and MDA-MB-231/HAS2 (HA+2), enabling these cells to form substantial pericellular matrices. NK-cell/trastuzumab/cetuximab-mediated ADCC is diminished in both of the HAS2-transfected cell types, suggesting that HA accumulation may contribute to tumor cell resistance to ADCC. Similarly, when these cells are treated in vitro with PEGPH20, a pegylated PH20 hyaluronidase (3), sensitivity to trastuzumab/cetuximab-mediated ADCC of the HAS2-overexpressing tumor cells is increased by up to 2-fold. These experiments show that the pericellular matrix coat (“turtle shell”) of high HA-producing tumor cells can inhibit the access of human immune cells to target cells, and that this effect can be reversed by depleting HA using PEGPH20. In vivo studies have shown that only PEGPH20, and not native PH20, has the bioavailability to induce antitumor activity in vivo. Models are currently in development to test whether PEGPH20 will enhance the activity of therapeutic MAbs in vivo.
Abstract B60: Overcoming drug resistant cancer by hyaluronic acid based nanogel formulated with anticancer drugs
Authors: Wei Xin , Senanayake Thulani H. , Vinogradov Serguei V.
Published: March 2013

One of major challenges of current anticancer therapies is the overcoming of emerging drug resistances and the preventing of tumor relapse. Among different causes of tumor resistance to therapy, the most important is probably the presence of a population of cancer initiating/stem cells (CSC) distinguished from other cancer cells by low metabolic activity, protective apoptotic signaling pathways and overexpression of drug efflux transporters [1, 2]. Many drug-resistant tumors and CSC display the overexpressed levels of CD44 receptor, a cellular glycoprotein binding hyaluronic acid (HA) [3]. The HA-CD44 interaction plays a prominent part in mediating cellular and intercellular events during cancer development. For biomedical applications, HA has been actively studied as a scaffold for tissue engineering and targeting moiety for drug delivery [4]. Here, we report an advanced design of HA-based polymeric nanogel-drug formulations with stronger activity compared to free drugs, which can potentially target CD44-expressing drug-resistant tumor cells.

The novel nanogels are based on the biocompatible and biodegradable HA-drug conjugates with a high drug load and linked hydrophobic moieties (e.g., cholesterol), which are capable of folding into nanogels in aqueous media. A set of nanogel conjugates of poorly soluble anticancer drug, including curcumin, etoposide and salinomycin, was obtained, characterized and studied in cancer cell cultures. Cholesterol is a common cellular component that was expected to facilitate nanogel-membrane interaction and the intracellular transport of these drugs. The drug content in nanogel conjugates was as high as 15–20% (NMR analysis). These nanogels formed very small and uniform particles of 30–40 nm in diameter as determined by dynamic light scattering. The small size of nanogels can facilitate passive tumor accumulation through leaky neovasculature (EPR effect) and drug distribution in tumor tissue. Cytotoxicity assays (MTT) demonstrated stronger anticancer activity of nanogel-drug conjugates compared to free drugs and HA-drug conjugates in human breast carcinoma cells in vitro. Fluorescent microscopy confirmed that these nanogels are internalized by receptor-mediated endocytosis depending on the expression of CD44 receptors on the surface of cancer cells. Potential further developments would include an optimization of the hydrophilic-lipophilic balance of these nanogel-drug conjugates, introduction of diagnostic and vector molecules, and the development of dual-drug nanoformulations. In conclusion, we developed and characterized in vitro a novel HA-based nanogel drug delivery and theranostic platform that exhibits high anticancer activity and has a potential to become a novel therapy for overcoming tumor drug resistance.
Abstract B63: Heterologous expression of serine palmitoyltransferase-1 alters chemosensitivity and multidrug resistance profile of human cancer cells of different tissue origins
Authors: Yerokun Tokunbo
Published: March 2013

The progression of human brain, bladder and prostate cancers, considered inflammatory cancers, is characterized by the acquisition of metastatic and drug resistance profiles which remain major impediments to the efficacious delivery of therapeutic intervention. Recent localization and protein interaction studies of the sphingolipid metabolism protein, serine palmitoyltransferase-1, SPTLC1, suggest a signaling, other than metabolic role during cellular response to diverse stress stimuli. The current study examined chemosensitivity to genotoxic compounds by a set of SPTLC1 recombinant cancer cell lines. When exposed to the anti-cancer drugs celecoxib and fenretinide, SPTLC1 localization as determined by confocal immunofluorescence microscopy showed its translocation to focal adhesion sites. This allows for possible interaction and crosstalk with other stress response proteins involved in regulating cell growth, differentiation, proliferation, apoptosis, migration and other processes by which cells respond to stress stimuli. Stress-inducible gene expression profile of cytochrome p450 transcripts shows differential attenuation of the Cyp1A1, 1A2 and 3A4 species. Altered expression of the genes for the cytochrome p450 xenobiotic metabolism proteins can have profound effect on the toxicity of anticancer drugs especially those with narrow therapeutic window. Taken together, the heterologous expression of a C-terminal modified SPTLC1 gene in the human cancer cell lines used in this study was found to mediate an altered drug response profile consistent with acquisition of a multidrug resistance phenotype. This is exemplified in increased resistance to anti-proliferative effect of therapeutic drugs, differential attenuation of transcriptional expression of Cyp450 species, as well as, chemical induced DNA fragmentation. The observations further support a general role for SPTLC1 in cellular stress response and its potential contribution in cellular resistance to cytotoxic drugs.
Abstract B7: Annexin V inhibits local but not systemic murine fibrosarcoma growth by blocking phosphatidylserine
Authors: Masadeh Maen , Yin Chaobo , Shilyansky Joel
Published: March 2013

Introduction: Phosphatidylserine (PS), a phospholipid expressed on the cytoplasmic surface of plasma membrane, becomes externalized during apoptosis and regulates immune activation. During our previous studies, we found that live tumor cells can express PS on the cell surface. We hypothesized that PS expression allows tumors to escape immune destruction, and blocking PS with annexin V (AnV) would retard tumor growth.

Materials and methods: CMS5 fibrosarcoma tumor cells were engineered to secrete AnV. Balb/c mice were inoculated subcutaneously with 105-106 CMS5 cells. Tumor growth was determined. Data was analyzed using student t-test.

Results: WT tumors grew significantly faster than AnV secreting tumors, with average size of 0.876 cm3 compared to 0.320 cm3 (p<0.05) in immune competent but not in SCID mice. To test whether AnV secreting tumors are intrinsically altered we inoculated mice with a mixture of 25% AnV secreting and 75% WT tumor cells. Growth of mixed cell tumors was identical to AnV secreting tumors, and was significantly smaller than WT tumors, 0.876 cm3 compared to 0.324 cm3 (p<0.05). To determine whether the effect of AnV was local or systemic mice were inoculated with tumor cells on each flank. Regardless of the type of tumor on the opposite side, WT tumors grew significantly faster than AnV secreting tumors with average size of .684 cm3 versus 0.345 cm3 (p<0.05).

Conclusion: We demonstrated blocking tumor PS with AnV retards local tumor growth. The effects of AnV require an intact immune system. These findings suggest that PS promotes tumor tolerance and blocking PS with AnV may be used in future immunotherapy studies.
Abstract IA1: Keynote Presentation: BRAF and RAS signaling in melanoma from basic to translational and clinical research
Authors: Marais Richard M.
Published: March 2013

BRAF is a protein kinase that is mutated in about half of human melanomas. Its activator, the small G-protein NRAS, is mutated in another 20% of cases. Drugs that inhibit BRAF mediate impressive responses in melanoma patients whose tumors carry mutations in BRAF, but are not effective in patients whose tumors express wild-type BRAF. Unexpectedly however, whereas BRAF drugs inhibit MEK/ERK signaling in melanoma cells that express oncogenic BRAF, they activate MEK/ERK signaling in cells that express oncogenic RAS. This paradoxical activation of MEK/ERK signaling in melanoma cells that express oncogenic RAS occurs because BRAF drugs drive the formation of dimers between BRAF and a closely related family member, called CRAF. These hetero- and homo-dimers contain one partner that is drug-bound and one partner that is drug-free, and the drug-bound partner activates the drug-free partner through a conformational change, or by acting as a scaffold protein. Notably, this paradoxical activation drives the formation of non-melanoma skin lesions (keratoacanthomas, squamous cell carcinomas) in about a third of patients. Using a two-stage skin carcinogenesis model, we find that this is not because BRAF inhibitors are tumor promoters per se; rather they act by accelerating the inevitable in susceptible patients by driving the growth of pre-existing lesions through paradoxical RAF activation. We also find that nilotinib is a weak RAF inhibitor that activates the RAF paradox and thereby drives survival in drug-resistant chronic mylogenous leukemia (CML) cells. Our data show how improved understanding of signaling responses to BRAF inhibitors can lead to improved patient care.
Abstract IA7: Novel mechanisms of resistance in BRAF-mutant melanoma
Authors: Poulikakos Poulikos I.
Published: March 2013

Tumors with mutant BRAF(V600E) are dependent on ERK signaling for their growth. Unlike MEK inhibitors that suppress ERK signaling in all cells, RAF inhibitors suppress ERK signaling in cells with mutant BRAF, but paradoxically enhance ERK signaling in tumor and normal cells with wild-type BRAF. The mechanistic basis of this phenomenon lies in the interaction of these drugs with RAF dimers. Active RAS promotes homo- and heterodimerization of RAF. Binding of drug to one protomer in the RAF dimer transactivates the other. In BRAF(V600E) tumors, RAS is not activated, RAF is a monomer and thus transactivation is minimal. In these tumors, RAF inhibitors bind to and inhibit the RAF monomer and effectively suppress ERK signaling. Furthermore, because RAF inhibitors do not inhibit ERK signaling in other cells, the model predicts that they would have a higher therapeutic index and greater antitumor activity than MEK inhibitors. Indeed, RAF inhibitors have shown remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E), however, resistance invariably develops. The mechanism of action of these drugs suggests that drug resistance could result from any lesion that increases RAF dimerization. These could include mechanisms that increase RAS activation in the tumor cells or lesions that cause RAF to dimerize in a RAF-independent manner. Indeed, mutations in NRAS and activation of receptor tyrosine kinases have now been associated with innate or acquired resistance to RAF inhibitors. We have found that aberrantly spliced forms of BRAF(V600E) are associated with resistance to RAF inhibitors in preclinical models and in patients that relapsed on the RAF inhibitor vemurafenib (PLX4032). These variants lack the RAS-binding domain, constitutively homodimerize in cells with low levels of RAS activity and confer resistance to the drug. Thus, RAF dimerization emerges as a common theme in RAF-inhibitor resistance mechanisms. Since a large part of resistance to RAF inhibitors is attributable to attenuation of the ability of the drug to inhibit RAF, one would predict that such tumors would retain at least partial sensitivity to inhibitors of downstream effectors of RAF such as MEK or ERK. Therefore, combinatorial approaches targeting upstream or downstream of RAF in addition to RAF inhibitors may delay or prevent the onset of these mechanisms of resistance.
Abstract IA9: The MEK network as a target for combination therapies
Authors: Korn W. Michael
Published: March 2013

Small molecule inhibitors of signal transduction pathways can elicit complex and sometimes non-intuitive molecular responses in cancer cells. Increasing evidence supports the notion that these responses are important mediators of de novo drug resistance. We systematically assessed molecular and cellular responses to MEK inhibitors, aiming at identifying meaningful targets for combination therapies. Proteomic analyses revealed an EGFR-dependent feedback loop resulting in activation of the PI3 kinase pathway following MEK inhibition. In agreement with this, combinations of MEK inhibitors and inhibitors of EGFR inhibitors or PI3 kinase show synergistic effects on cell proliferation and apoptosis rates in molecularly defined disease subtypes. To further explore molecular networks affected by MEK inhibition, Bayesian network inference algorithms were applied and reverse-engineered networks were generated that are amenable to in silico forward-simulation. Using this technology, new, MEK-dependent, novel regulators of cell cycle and apoptosis were identified and experimentally validated. These findings highlight the potential of mathematical modeling approaches to efficiently reveal new and therapeutically relevant features of signal transduction networks.
Abstract PR2: Stroma-derived matrix metalloproteinase 9 (MMP9) confers resistance to anti-EGFR therapy through cell-extrinsic activation of ERBB2/ERK/JUN pathway
Authors: Woo Janghee , Teppei Morikawa , Ogino Shuji , Shivdasani Ramesh A.
Published: March 2013

Matrix metalloproteinases (MMPs) have been studied historically for their role in tissue invasion and metastasis. The tumor microenvironment also produces MMPs that may influence other tumor behaviors, as recent mouse models suggest in the intestine and other sites. We examined MMP9 expression in 642 cases of human colorectal cancer (CRC) in relation to clinicopathological features and mutations. MMP9 antibody (Ab) unambiguously stained mesenchymal cells in 104 cases (16.2%) and gave weaker stromal staining in another 12.8%. High stromal MMP9 expression correlated most strongly with the CpG island methylator phenotype (CIMP-high) in tumors (multivariate odds ratio – OR – 2.45, 95% confidence interval – CI – 1.41 to 4.27, p=0.0015) and also with absence of tumor KRAS mutations (multivariate OR 0.54, 95% CI 0.33 to 0.87, p=0.011). The latter association suggested KRAS allelic status as one reason for heterogeneity in CRC cell line responses to MMP9. Four out of 6 KRASWT cell lines proliferated significantly more in the presence of exogenous MMP9, while 4 of 5 KRASMut lines failed to respond. This led us to ask if stromal MMP9 expression might predict the outcome of treatment with anti-EGFR mAb cetuximab, allowing KRASWT tumors to bypass mAb blockade of EGFR-KRAS pathway activation. We retrospectively evaluated MMP9 expression by immunohistochemistry on primary tumors and progression-free survival (PFS) in 86 patients who had received chemotherapy combined with cetuximab as first-line or salvage therapy. Tumors from 29 patients (33.8%) harbored mutant KRAS. PFS of patients with MMP9high;KRASWT tumors (median 3.5 months) was lower than that of patients with low or no stromal MMP9 expression and KRASWT tumors (median 5.8 months, 95% CI for the hazard ratio 1.135-2.179). Patients with KRASMut tumors showed no significant difference in PFS with respect to MMP9 expression (2.55 months and 2.25 months in the MMP9high and MMP9neg/low subgroups, respectively). Thus, low stromal MMP9 expression in KRASWT tumors predicted a better outcome with cetuximab treatment than high MMP9 expression. In CRC cell lines that grow faster in response to MMP9, this factor activates ERBB2 and ERK/JUN signaling. Together, these findings suggest that stroma-derived MMP9 may help tumors bypass common mutational mechanisms for constitutive growth factor pathway activation and confer resistance to anti-EGFR therapy through cell-extrinsic activation of the ERBB2/ERK/JUN pathway. Stromal MMP9 expression may thus predict cetuximab response in KRASWT CRCs and have value in selecting patients for treatment.

This proffered talk is also presented as Poster A26.
Abstract PR5: Identifying mechanisms of drug resistance to MAPK pathway inhibition via genome-scale rescue screens
Authors: Johannessen Cory M. , Johnson Laura A. , Lam Daniel , Hamm Luke , Piccioni Federica , Root David , Garraway Levi
Published: March 2013

The recent clinical success of RAF inhibitors in BRAF(V600E)-mutant malignant melanoma highlights the promise of targeted therapeutics as well as the inevitability of acquired drug resistance: responses are sustained for less than a year and all patients ultimately become drug resistant. To identify causative drug resistance genes in BRAF(V600E)-mutant melanoma, we have individually expressed over 14, 000 unique genes and queried their ability to affect sensitivity to a panel of mitogen activated protein kinase (MAPK) pathway inhibitors, including a RAF, MEK and ERK inhibitor, both as single agents or in combination (RAFi+MEKi). Out of this genome-scale collection, less than 1% of genes are sufficient to induce resistance to the MAPK pathway inhibitors tested. Here, we present the results of this rescue screen and extensive validation/characterization efforts that reveal a number of novel insights into mechanisms of drug resistance, melanoma biology and the MAPK signaling pathway. Additionally, the scale of this first-of-kind functional rescue screen permits its integration with existing genome characterization efforts, including melanoma sequencing studies, gene expression data and parallel functional assays to identify the most relevant genetic modifiers of resistance to MAPK pathway inhibition.

This proffered talk is also presented as Poster B27.