bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2022: Twist1 regulates UVB-induced epidermal cell proliferation in non-melanoma skin cancer
Authors: Srivastava Jaya , Rho Okkyung , DiGiovanni. John
Published: July 2016

Nonmelanoma skin cancer (NMSC) is the most common cancer and consists of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). SCC metastases account for up to 20% of all deaths from skin cancer. The major risk factor for the development of NMSC is solar ultraviolet radiation (UV). UVA and UVB both function as initiators and promoters in carcinogenesis and possess immunosuppressive activity. Given the prevalence of this disease, there remains a pressing need for the development of novel prevention and treatment strategies for NMSC.

In a recent study (Srivastava et al. 2015. Mol. Carcinog. doi:10.1002/mc.22335), we evaluated the effect of Twist1, a transcription factor that is known to play a role in tumor progression, during epithelial carcinogenesis. Using the two-stage chemical carcinogenesis model, we identified that BK5.Cre x Twist1flox/flox mice, which have a keratinocyte-specific Twist1 knockout (Twist1 KO), developed significantly reduced numbers of papillomas (70% reduction) and SCCs (75% reduction) as well as delayed tumor latency compared to wild-type (WT) mice. Twist1 KO mice also exhibited significantly reduced epidermal proliferation in response to chemical promotion that correlated with reduced levels of cell cycle regulators and increased levels of p53 and p21.

In further studies, we have begun analyzing the effects of UVB irradiation in Twist1 KO mice. Specifically, Twist1 KO mice exhibited heightened sensitivity to UVB-induced apoptosis and decreased epidermal proliferation as compared to similarly exposed WT mice. In addition, the accumulation of mast cells in the skin was significantly decreased in Twist1 KO mice as compared to WT mice. Further preliminary experiments showed that the protein expression levels of c-Myc, phospho-p65 (S536) and phospho-p50 (S337) decreased following UVB irradiation whereas levels of the cell cycle inhibitors p21 and p27 exhibited an increased expression in Twist1 KO mice as compared to WT mice. Further experiments have suggested that Twist1 may play a pivotal role in the proliferation/migration of bulge region keratinocyte stem cells (KSCs) following exposure to UVB. These and other results from ongoing experiments will be presented. Collectively, these findings suggest that Twist1 may have a novel role in UVB-induced epithelial carcinogenesis by regulating proliferation and migration of keratinocytes and KSCs. Research supported by CPRIT RP150346, CPRIT RP140108, and NIH T32 ES007247.

Citation Format: Jaya Srivastava, Okkyung Rho, John DiGiovanni. Twist1 regulates UVB-induced epidermal cell proliferation in non-melanoma skin cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2022.
Abstract 2024: BP1 homeoprotein induces tumor cell growth and estrogen-independent tumorigenesis in mice
Authors: Fu Sidney W. , Kirolikar Saurabh , Ginsburg Erika , Schwartz Arnold , Simmens Samuel , Berg Patricia E.
Published: July 2016

BACKGROUND. BP1 is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 overexpression increases cell survival, aggressiveness and metastasis in breast cancer cells. BP1 protein is expressed in 80% of invasive ductal breast tumors, including 100% of ER negative (ER-) tumors and 73% of ER positive (ER+) tumors. Therefore we hypothesize that BP1 may regulate ER in breast cancer. A mouse model was developed to test the regulatory effects of BP1 in ER expression. MATERIALS AND METHODS. MCF-7 breast cancer cells transfected with BP1 or vector control were njected into the fat pads of nude mice. Regulation of ER by BP1 was studied using ChIP assays; EMSA assays were used to test binding of BP1 to ER DNA. Tamoxifen assays were used to evaluate the effects of BP1 overexpression on tamoxifen resistance. RESULTS. MCF-7 cells overexpressing BP1 or control cells containing an empty vector were injected into nude mice. Approximately half of the estrogen-supplemented mice developed tumors. Although MCF-7 cells require exogenous estrogen to form tumors in nude mice, approximately 20% of the mice implanted with cells overexpressing BP1 were able to form tumors in the absence of added estrogen. To understand the molecular mechanisms of this observation, we carried out in vitro experiments. Our data suggest that BP1 regulates ER both directly and indirectly: (1) Direct: ChIP assays and EMSA assays showed that BP1 binds to the first intron of the ER alpha gene and transcriptionally activates it, leading to increased ER protein. (2) Indirect: regulation of ER also occurs indirectly by BP1 upregulation of p300, an activator of ER, and also by repression of BRCA1 by BP1; BRCA1 destabilizes ER protein. Furthermore, MCF-7 cells overexpressing BP1 are more resistant to growth in 3uM tamoxifen compared with empty vector containing cells. CONCLUSIONS. BP1 overexpression in MCF-7 cells injected into mice not only increases tumor size but also promotes estrogen-independent tumorigenesis, and leads to resistance to tamoxifen. Therefore, BP1 could serve as a novel therapeutic target for ER+ breast cancer.

Citation Format: Sidney W. Fu, Saurabh Kirolikar, Erika Ginsburg, Arnold Schwartz, Samuel Simmens, Patricia E. Berg. BP1 homeoprotein induces tumor cell growth and estrogen-independent tumorigenesis in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2024.
Abstract 2026: DEC1 negatively regulates AMPK activity via LKB1
Authors: Sato Fuyuki , Muragaki Yasuteru
Published: July 2016

Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in metabolism and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, how the circadian rhythm of AMPK activity is regulated is not known. The aim of this study is the direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. A medium change and serum shock led to rhythmic patterns of DEC1 protein and AMPK activity levels in WI-38 cells, and their peak levels occurred at different times. However, changing the medium did not induce circadian rhythms of DEC1 protein or AMPK activity levels in MCF-7 and U2OS cells, in which the intensities of these levels had inversely correlated patterns. Knocking down DEC1 expression increased AMPK activity, whereas DEC1 overexpression decreased it. DEC1 bound to the E-box of the LKB1 promoter, decreasing the LKB1 activity and total protein levels. In addition, knocking down DEC1 expression inhibited cell death under the condition of glucose depletion, increasing the activity of both AMPK and LKB1. Taken together, our results showed that the levels of DEC1 protein and AMPK activity were affected even long after the medium change and that the rhythmic patterns of these levels were inversely correlated. We concluded that DEC1 negatively regulated AMPK activity via LKB1.

Citation Format: Fuyuki Sato, Yasuteru Muragaki. DEC1 negatively regulates AMPK activity via LKB1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2026.
Abstract 205: Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay
Authors: Chen Lu , Saporita Anthony J. , Lie Wen-Rong , Maheshwari Reeti , Schluter Melissa , Mistry Jehangir , Hwang Joseph
Published: July 2016

A novel multiplex immunoassay was developed for examining acquired resistance to Receptor Tyrosine Kinase (RTK) inhibitors. RTKs are transmembrane proteins which act as receptors for growth factors, neurotrophic factors and other extracellular signaling molecules. These cell-surface kinases are activated by extracellular ligands leading to receptor dimerization and tyrosine phosphorylation at specific residues in the cytoplasmic tails to initiate RTK-mediated signal transduction. Of the >500 known protein kinases in the human genome there are approximately 60 RTKs. They are central components of cell signaling networks and play crucial roles in normal physiological processes and disease processes ranging from diabetes to cancer. Many RTKs, such as EGFR, HER2, c-Kit, PDGFR and VEGFR, have been used as targets for drug development and RTK-targeted therapies have illustrated the utility of these treatments for selected cancers. However, in many cases, compensatory RTK signaling enables cancer cells to acquire resistance to RTK inhibitors that selectively target a single RTK. For example, some EGFR and HER2 inhibitors have led to resistance and are associated with increased expression of IGF1R. In order to understand the mechanisms of resistance to RTK inhibitors, we have developed a bead-based multiplex immunoassay capable of simultaneously detecting phosphorylation of 18 different RTKs. First we demonstrate detection of RTK phosphorylation in response to growth factor stimulation using various cell lines. Next we examined the specificity of two inhibitors targeting EGFR and HER2. These inhibitors specifically reduced growth factor-stimulated phosphorylation of EGFR and HER2, without inhibiting the activation of other RTKs. Although we did not observe compensatory activation of other RTKs in response to the two EGFR and HER2 inhibitors we tested, this study demonstrates the feasibility of using this novel 18-plex RTK panel for examining the mechanism of resistance to single RTK inhibitors. In summary, the RTK multiplex panel allowed for simultaneous detection of multiple tyrosine phosphorylated RTKs in a specific, sensitive, and reproducible manner.

Citation Format: Lu Chen, Anthony J. Saporita, Wen-Rong Lie, Reeti Maheshwari, Melissa Schluter, Jehangir Mistry, Joseph Hwang. Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 205.
Abstract 2053: Effects of thymoquinone encapsulation on its uptake, delivery and anticancer activity in breast cancer cells
Authors: Fakhoury Isabelle , Saad Walid , Bou Hadir Kamal , Schneider-Stock Regine , Gali-Muhtasib Hala
Published: July 2016

Introduction: Thymoquinone (TQ) is a promising anticancer molecule which suffers from limited bioavailability. Drug encapsulation is commonly used to overcome low drug solubility, bioavailability as well as nonspecific targeting. For this project, we synthesized different TQ nanoparticles (TQ-NPs), characterized their properties, uptake and delivery mechanisms, and assessed their anticancer potential in a panel of breast cancer cells.

Methods: The different TQ-NPs were prepared using Flash nanoprecipitation. Dynamic light scattering and scanning electron microscope were used for the characterization of the size, morphology and stability of the NPs. The anticancer effect was assessed by MTT. We subsequently formulated fluorescent TQ-NPs and evaluated their uptake and subcellular intake mechanism by both fluorometry and confocal microscopy.

Results: We were successful at formulating four different stable TQ-NPs that had an average diameter size between 45-130 nm. All TQ-NPs had also high entrapment efficiency and loading content. In vitro, TQ-NPs enhanced the antitumor activity of the drug in both MCF-7 and MDA-MB-231 cells, when compared to free TQ with no significant cytotoxicity of the blank NPs. Fluorescent TQ-NPs uptake was further found to be both time and concentration dependent. Finally, using inhibitors of endocytosis we determined that the endocytosis of TQ-NPs is caveolin mediated. This was also confirmed by examining the subcellular localization of TQ-NPs. The nanoparticle formulations colocalized with both caveolin and transferrin but not with lamp-1 and EEA-1 proteins.

Conclusion: Altogether, our results describe a new approach for the enhancement of TQ anticancer activity and provide insights on the uptake dynamics specific to our formulation.

Citation Format: Isabelle Fakhoury, Walid Saad, Kamal Bou Hadir, Regine Schneider-Stock, Hala Gali-Muhtasib. Effects of thymoquinone encapsulation on its uptake, delivery and anticancer activity in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2053.
Abstract 2061: DK049, a novel acid-sensitive prodrug of gemcitabine: design, in vitro properties and in vivo efficacy
Authors: Koester David , Garcia Javier , Fuchs Olga , Kratz Felix
Published: July 2016

Gemcitabine (Gemzar®) is an anticancer nucleoside that is widely used to treat solid tumors. Unfortunately, at its recommended dose of ∼1000 mg/m2 approximately 90% are deactivated by cytidine deaminase to the inactive uridine metabolite and excreted in the urine. A further disadvantage resulting in chemo-resistance is the low expression level of the human equilibrative nucleoside transporter 1 (hENT1) on the cell surface of cancer cells thus preventing substantial uptake of gemcitabine. In order to circumvent these disadvantages, we designed an innovative gemcitabine prodrug DK049 that a.) is capped at the amino group of the cytosine moiety with acetyl benzoic acid preventing deamination, and b.) incorporates a proprietary maleimide-bearing LADR™ linker forming a stable, but acid-sensitive hydrazone bond. At its 5′-OH position a phosphate group was introduced rendering water-solubility. Following intravenous administration, DK049 binds rapidly and selectively to circulating albumin and utilizes the advantages of albumin-mediated tumor uptake as well as cellular uptake by endocytosis (1).

After cleavage of the acid-sensitive benzoyl hydrazone bond, we could show that the acetyl benzoic acid is decapped by carboxylesterase 2. In a head-to-head comparison with gemcitabine (4 × 240 mg/kg on a weekly basis) in four subcutaneous human patient-derived tumor xenografts (NSCLC: LXFE397, LXFE937, ovarian cancer: OVXF899, and pancreatic cancer: Panc11159; 8-10 nude mice per group), DK049 (4 × 24 mg/kg gemcitabine equivalents or 8 × 18 mg/kg gemcitabine equivalents over 4 weeks) demonstrated distinctly superior antitumor efficacy versus gemcitabine (p < 0.05). In the LXFE937 and OVXF899 model, DK049 induced long-term significant regressions for several weeks after end of therapy without any loss in body weight or bone marrow toxicity. In summary, these results show a superior long-term antitumor efficacy of DK049 compared to gemcitabine at a significantly reduced dose.

(1) Felix Kratz, Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release 8;132:171-83, 2008.

Citation Format: David Koester, Javier Garcia, Olga Fuchs, Felix Kratz. DK049, a novel acid-sensitive prodrug of gemcitabine: design, in vitro properties and in vivo efficacy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2061.
Abstract 2062: Nanoemulsion formulations for anticancer agent piplartine characterization, toxicological, pharmacokinetics and efficacy studies
Authors: Fofaria Neel , Ranjan Alok , Qhattal Hussaini Syed Sha , Liu Xinli , Srivastava Sanjay K.
Published: July 2016

Piplartine (PL) is an alkaloid found in black-pepper and known for its anticancer activity, however, due to poor solubility and lack of proper formulation, its use for oral

administration is a challenge. The objective of this study was to formulate PL into nanoemulsion drug delivery system for oral delivery and thereafter evaluate toxicity, pharmacokinetics and therapeutic efficacy. Optimized nanoemulsions were formulated by self-emulsification as well as by homogenization-sonication method. Two nanoemulsions enhanced the solubility of PL with low polydispersity index and high stability. Both PL loaded nanoemulsions exhibited enhanced dissolution, cellular permeability and cytotoxic effects as compared to pure PL. Formulation of PL into nanoemulsions did not obstruct its cellular uptake in cancer cells. Blank or PL loaded nanoemulsions did not exhibited toxicity in mice upon daily oral administration for 60 days. Pharmacokinetics of PL followed a two-compartment model after intravenous administration. PL loaded nanoemulsions showed 1.5-fold increase in oral bioavailability as compared to free PL. Finally, PL loaded nanoemulsions showed marked anti-tumor activity at a dose of 10 mg/kg in melanoma tumor bearing mice. In conclusion, for the first time we have developed a stable nanoemulsion delivery system for oral administration of PL, which enhanced its solubility, oral bioavailability and anti-tumor efficacy.

Citation Format: Neel Fofaria, Alok Ranjan, Hussaini Syed Sha Qhattal, Xinli Liu, Sanjay K. Srivastava. Nanoemulsion formulations for anticancer agent piplartine characterization, toxicological, pharmacokinetics and efficacy studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2062.
Abstract 2079: IONP-LPrA2 and PEG-LPrA2 therapies for triple negative breast cancer
Authors: Harmon Tia L. , Harbuzariu Adriana , Rampoldi Antonio , Dill Courtney , Lanier Viola , Daley-Brown Danielle , Lipsey Crystal , Tchio Cynthia , Yang Lily , Gonzalez-Perez Ruben Rene
Published: July 2016

Background: Breast cancer (BC) is an epidemic in the US. It is estimated that there will be over 230,000 new BC diagnoses in 2016. Triple Negative Breast Cancer (TNBC) comprises ∼15% of BC cases and lacks targeted therapeutic options. Obesity and high leptin levels are associated with higher TNBC incidence and poorer patient outcomes. Overexpression of leptin and its receptor, Ob-R, induce BC cell growth and angiogenesis; therefore leptin/Ob-R may serve as a TNBC therapeutic target. We have developed a Leptin Peptide Receptor Antagonist, LPrA2, which effectively inhibits leptin signaling. To increase its efficacy LPrA2

was coupled to iron oxide nanoparticles (IONPs) and polyethylene glycol (PEG), and subsequently tested in vitro and in vivo in obese mice hosting syngeneic TNBC-like mammary tumors.

Methods: The conjugation of LPrA2 to IONPs and PEG was confirmed by immunoblotting analysis. E0771, mouse BC cells, which are progesterone receptor and HER2 negative were made insensitive to estrogen stimuli by long-term treatment with Tamoxifen (TAM). Cell cycle and MTT assays were performed to determine the effectiveness of leptin signaling inhibition by conjugated LPrA2 in vitro. C57BL/6 female mice were fed a 60% fat diet

to induce obesity. The obese mice were injected with E0771-TAM cells in the mammary fat pad and treated with IONP-LPrA2 and PEG-LPrA2 after tumor development. Obese mice treated with IONP-LPrA2 Scramble (Sc) and PEG-LPrA2 Sc served as negative controls.

Results and Conclusion: IONP-LPrA2 and PEG-LPrA2 attenuated leptin signaling in vitro and decreased tumor growth and progression in vivo in comparison to the controls. These findings indicate that conjugated LPrA2

may serve as a targeted therapy for TNBC. IONP-LPrA2 may be especially useful in treating this more aggressive form of BC due to its ability to capture multiple LPrA2 peptides as well as its small and uniform particle size.

Citation Format: Tia L. Harmon, Adriana Harbuzariu, Antonio Rampoldi, Courtney Dill, Viola Lanier, Danielle Daley-Brown, Crystal Lipsey, Cynthia Tchio, Lily Yang, Ruben Rene Gonzalez-Perez. IONP-LPrA2 and PEG-LPrA2 therapies for triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2079.
Abstract 2086: Noscapine chemosensitization enhances docetaxel anticancer activity and tumor stroma disruption against triple negative breast cancer
Authors: Doddapaneni Ravi , Patel Ketan , Chowdhury Nusrat , Sachdeva Mandip
Published: July 2016

Purpose:

The use of Noscapine (Nos) as a chemosensitizer followed by docetaxel (DTX) treatment therapy could be a novel approach for the treatment for breast cancer and possibly reduce the adverse side effects associated with DTX based chemotherapy. The goal of this study was to examine the chemo-sensitizing effect of Nos to DTX and also tumor stromal disruption effect of Nos in mice bearing xenograft TNBC tumors.

Methods:

Effect of Nos chemosensitization on DTX cytotoxicity was evaluated in MDA-MB-231 cells by trypan blue dye method. Apoptosis was measured by AnnexinV/FITC method using flow cytometer. Expression of different proteins like phospho-p38, pJNK, bcl-2, α-tubulin, Akt, pAkt, survivin was evaluated by immunoblot. Alpha-tubulin binding assay was done by fluorescent microscopy. In vivo antifibrotic efficacy of Nos and uptake of coumarin-6 loaded fluorescent liposomes was evaluated by picro-sirius red staining and fluorescent microscopy respectively in xenograft breast tumors.

Results:

MDA-MB-231 TNBC cells were exposed at sub-therapeutic dose of Nos (4 μM) which increased the cytotoxicity of DTX by 3.0-fold. Flow-cytometric analysis showed significant increase (30 percent) of late apoptotic cells in Nos chemosensitized, DTX-treated MDA-MB-231 cells compared with DTX alone treatment. Further, chemosensitization of TNBC cells with Nos at different time intervals (6 h, 12 h and 24 h), the effect on stress transducer p38 stress activator protein kinase was significantly activated (p<0.01). Also, Nos (4 μM) chemosensitized cells followed by DTX treatment showed downregulation of bcl2 (2.3-fold), survivin (1.3-fold) and pAKT (1.9-fold) expression further illustrating the role of Nos in inducing apoptosis. Interestingly, at this dose of Nos (4 μM), there was no impact on alpha-tubulin. Also, in vivo studies with 100 mg/kg Nos given orally to MDA-MB-231 tumor bearing athymic nude mice revealed significant reduction in tumor collagen-1 levels in Nos treated group compared to control as determined by picro-sirius staining. In Nos treated tumors, uptake of coumarin-6 loaded fluorescent liposomes was 7-fold higher compared to controls suggesting antifibrotic role of Nos.

Conclusion:

In conclusion, chemosensitization with sub-therapeutic dose of oral noscapine could be a promising approach to increase anticancer activity of DTX and can further enhance uptake of liposomes suggesting that this approach may have potential in TNBC treatment.

Citation Format: Ravi Doddapaneni, Ketan Patel, Nusrat Chowdhury, Mandip Sachdeva. Noscapine chemosensitization enhances docetaxel anticancer activity and tumor stroma disruption against triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2086.
Abstract 2095: Toxicity of selective allosteric FGFR2 inhibition in preclinical studies
Authors: Lapina Nataliya V. , Melikhova Marina V. , Vakunenkova Olga A. , Kashuro Vadim A.
Published: July 2016

Alofanib (RPT835, Ruspharmtech LLC) is a novel selective allosteric inhibitor binding to the extracellular domain of FGFR2. Alofanib had no direct effect on activation of FGFR1 and FGFR3 in vitro. Selective effect of the compound may reduce toxicity compared to multi-targeted VEGFR/FGFR or pan-FGFR inhibitors. This study characterizes the organ toxicity of alofanib in Sprague-Dawley rats and Chinchilla rabbits, and the reversibility of any treatment-induced effects.

240 rats and 30 rabbits received alofanib (0-40.5 and 0-21.6 mg/kg/day, respectively) intravenously on a consecutive daily dosing schedule for thirteen weeks. Clinical observations and laboratory parameters were recorded. Necropsy was conducted following treatment/recovery periods, and histologic examinations were performed.

There was no treatment-related mortality, severe toxicity and significant changes in organs in the thirteen-week study. Alofanib was well-tolerated in vivo. Clinical signs of first injection (8.1 mg/kg) included moderate local irritative reaction and decreased activity during hour in rats. Clinical symptoms of toxicity were not observed over study. Animals were active. Body weight remained stable and it was comparable to vehicle groups. Abnormal electrocardiogram findings and blood pressure changes were not detected. It was notable that in the study, none of the hematologic changes was present at the end of the recovery period. Minimal significant decrease in ALT/AST, slight increase in alkaline phosphatase in male rats and mild increase in urea, decrease in chlorine in female rats were found throughout treatment period. Minimal significant decrease in albumin and chlorine was identified in rabbits. Rate of hyperphosphatemia, an on-target side-effect of pan-FGFR inhibitors, will be presented at the AACR Meeting.

We conclude that toxicity of alofanib was mild in the thirteen-week study. Slight decrease of chlorine in blood was most common side effect. The final conclusion will be made after six-month study. This study was supported by a grant from the Skolkovo Foundation.

Citation Format: Nataliya V. Lapina, Marina V. Melikhova, Olga A. Vakunenkova, Vadim A. Kashuro. Toxicity of selective allosteric FGFR2 inhibition in preclinical studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2095.
Abstract 2102A: Simultaneous targeting of EGFR and ALK in EML4-ALK positive lung cancer to inhibit tumor cell proliferation and migration
Authors: Li Chunrong , Kern Joseph G. , Huang Shyhmin , Armstrong Eric A. , Werner Lauryn R. , Harari Paul M.
Published: July 2016

Approximately 4-7% of non-small cell lung cancers (NSCLC) harbor the EML4-ALK chromosomal rearrangement. ALK has been identified as a molecular driver of cancer and ALK inhibitors have been developed for clinical use. We have confirmed high expression of EGFR in EML4-ALK-positive cell lines (H3122 and H2228). Recognizing the capacity of both EGFR and ALK signaling to regulate tumor cell growth, we investigated the effects of co-inhibition of ALK and EGFR on cell proliferation and migration in EML4-ALK-positive cells in vitro. We also examined the anti-tumor effect of ALK and EGFR inhibitors in xenograft mouse models. Cell proliferation was determined by CCK8 and cell migration was examined by wound healing assay and transwell migration assay. ALK inhibitors (crizotinb, ceritifinib and alectinib) and EGFR inhibitors (erlotinib and gefitinib) were utilized to block ALK and EGFR signaling activity.

We confirmed that H3122 and H2228 cells were resistant to EGFR inhibitors in comparison to EML4-ALK negative cell lines H226 and A549. Simultaneous knockdown of ALK and EGFR by siRNA significantly inhibited tumor cell proliferation. Combined inhibition of ALK and EGFR inhibited cell proliferation more potently than single inhibitor treatment alone. In addition, NIH3T3 cells expressing EML4-ALK variants 1, 2 or 3a displayed enhanced ability for cell migration, suggesting a role of EML4-ALK in cell migration. Individual ALK inhibitors and EGFR inhibitors inhibited cell migration. Further, combined ALK and EGFR blockade resulted in a stronger cell migration inhibition than single agent therapy. The combination of ALK inhibitor with EGFR inhibitor was more potent than single target treatment in tumor xenograft growth inhibition. Mechanistic analysis of the co-regulation of cell migration by ALK and EGFR signaling in EML4-ALK positive cells is under investigation.

These results identify enhanced inhibitory effects on tumor cell proliferation and migration with simultaneous blockade of ALK and EGFR signaling suggesting that dual targeting may prove more effective than single agent approaches. These findings indicate the potential for therapeutic benefit of co-targeting ALK and EGFR in patients with EML4-ALK positive NSCLC.

Citation Format: Chunrong Li, Joseph G. Kern, Shyhmin Huang, Eric A. Armstrong, Lauryn R. Werner, Paul M. Harari. Simultaneous targeting of EGFR and ALK in EML4-ALK positive lung cancer to inhibit tumor cell proliferation and migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2102A.
Abstract 2104: ERK-dependent IL-6 autocrine signaling mediates adaptive resistance to pan-PI3K inhibitor in head and neck squamous cell carcinoma
Authors: Yun Mi Ran , Kang Han Na , Cho Byoung Chul
Published: July 2016

Purpose: To investigate resistant mechanisms to NVP-BKM120, a pan-PI3K inhibitor, and potential combination therapies in head and neck squamous cell carcinoma (HNSCC).

Experimental Design: A panel of 10 human HNSCC cell lines including NVP-BKM120-resistant HNSCC patient-derived cell lines were used to evaluate the antitumor activity of NVP-BKM120. Patient-derived tumor xenograft (PDTX) models were established from recurrent/metastatic HNSCC patients treated with and resistant to NVP-BKM120.

Results: Treatment of NVP-BKM120 led to upregulation of interleukin 6 (IL-6) and subsequent activation of either extracellular signal-regulated kinase (ERK) or signal transducers and activators of transcription 3 (STAT3), resulting in a modest antitumor effects on HNSCC cells. In a subset of cell lines, blockade of IL-6 autocrine signaling with IL-6 receptor (IL-6R) siRNA or IL-6R neutralizing antibody completely abolished constitutive or NVP-BKM120-induced activation of ERK and STAT3 as well as expression of oncogenic factors including Bcl2 and cMYC, resulting in the enhanced sensitivity to NVP-BKM120 with a marked reduction of S phase and significant increment of G0/G1 arrest. Interestingly, trametinib, a MEK inhibitor, significantly reduced NVP-BKM120-induced IL-6 and cMYC upregulation, whereas it had no effect on STAT3 inhibition. Moreover, combination of NVP-BKM120 with trametinib yielded more potent anti-tumor effects than combination with a pharmacologic inhibitor of STAT3 or siSTAT3. Furthermore, combination of NVP-BKM120 with trametinib or tocilizumab, a humanised anti-IL-6 receptor antibody, synergistically suppressed the growth of NVP-BKM120-resistant PDTX models as compared with either agent alone, which was confirmed in two PDTX-derived cell lines.

Conclusions: Collectively, these results suggested that IL-6/ERK signaling contributes to intrinsic and adaptive resistance to NVP-BKM120 in HNSCC, providing a rationale for a novel therapeutic strategy to overcome resistance to pan-PI3K inhibitors.

Citation Format: Mi Ran Yun, Han Na Kang, Byoung Chul Cho. ERK-dependent IL-6 autocrine signaling mediates adaptive resistance to pan-PI3K inhibitor in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2104.
Abstract 2115: MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation
Authors: Kawakami Hisato , Huang Shengbing , Sinicrope Frank A.
Published: July 2016

Background. Oncogenic BRAFV600E mutations in colorectal cancer (CRC) are associated with poor prognosis and treatment resistance. BRAF mutations activate MAP kinase signaling and may confer apoptosis resistance by upregulating anti-apoptotic BCL-2/BCL-XL/MCL-1 proteins.

Results. Ectopic expression of mutant BRAFV600E was shown to potently activate MEK/ ERK that mediated the phosphorylation (T163) and upregulation of MCL-1, but not BCL-2 or BCL-XL. MEK/ERK-mediated phosphorylation of MCL-1 (T163) increased its protein stability, shown by an MCL-1 phospho-mimic mutant. In human colon cancers, mutant vs wild-type BRAF was associated with increased MCL-1 expression by IHC. MEK/ERK inhibition by cobimetinib induced pro-apoptotic BIM in BRAF mutant CRC cell lines (HT-29, RKO), and suppressed phospho-MCL-1/MCL-1 proteins as did ERK siRNA. Suppression of MCL-1 by shRNA or using a small molecule MCL-1 inhibitor (A-1210477) synergistically enhanced cobimetinib-induced apoptosis (Table) that was associated with BAK/BAX activation. A-1210477 antagonized MCL-1 by disrupting its interaction with BAK and BIM proteins, shown by immunoprecipitation.

Conclusion. BRAF/MEK/ERK induces upregulation of MCL-1 via phosphorylation/stabilization to confer apoptosis resistance that can be overcome by combined ERK and MCL-1 inhibition, suggesting a novel therapeutic strategy against BRAF mutant CRCs. Drug-induced apoptosis (annexin V) in CRC cell linesDrugsHT-29 % apoptosisRKO % apoptosisDMSO16.842.11A-1210477 (2.5 μM)29.5222.88Cobimetinib(5 μM)28.0421.05Combination53.2250.93

Citation Format: Hisato Kawakami, Shengbing Huang, Frank A. Sinicrope. MEK/ERK and MCL-1 inhibition synergistically reverse apoptosis resistance in colon cancer cells with BRAF(V600E)-mediated MCL-1-upregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2115.
Abstract 2121: Src mediates acquired resistance to ALK inhibitor in ALK-rearranged non-small cell lung cancers
Authors: Yoshida Ryohei , Okumura Shunsuke , Sasaki Takaaki , Osaki Yoshinobu
Published: July 2016

ALK tyrosine kinase inhibitors (TKIs) play a significant role in the treatment of ALK-rearranged non-small cell lung cancer patients.After the significant initial response to ALK-TKIs, the emergence of acquired resistance ultimately occur for all patients.

Understanding the origin of resistance mechanisms within a patient is crucial to guide treatment and developing drugs/combinations to circumvent resistance.

There are reported that some of the tyrosine kinases are involving by bypassing ALK signaling, but comprehensive identification of proteins that are significant for acquired resistance in ALK-TKIs is still required.

Quantitative proteomic analysis provides a powerful approach in screening for alterations in protein levels. An analysis of the differential proteins expression between original cancer cell and acquired resistance cell would be useful for identification of proteins drug resistance.

To investigate the changes of chaperon holding proteins, ALK rearranged-cell line H3122 were used. ALK-TKI Alectinib resistant cell lines were established by exposing increased dose of alectinib to ALK rearranged H3122 cell line (AFR). Luminespib (AUY922, Novartis Pharm.) was used as HSP90 inhibitor and Saracatinib (Selleck Chemicals.) was used as Src inhibitor in this study.

We used iTRAQ (isobaric tags for relative and absolute quantitation) technology using nano-LC-MS/MS analysis, to identify alterations in H3122 protein levels of pre/post HSP90 inhibitor treatment. For further analysis, protein expression was measured by western blot. To assess the cancer cell growth inhibition for ALK and/or Src inhibitors, we used colony formation assay.

Our results revealed that Crk-Src related proteins were overexpressed in ALK TKI resistance cells and had a significant protein expression changes after HSP90 inhibitor treatment. To address the impact of overcoming resistance by blocking Crk-Src pathway, Drugs/combinations with ALK inhibitor and/or Src inhibitors showed that significant treatment effect in vitro and in vivo study using tumor-bearing mouse model.

Conclusion

Src is one of the significant signaling pathways in the ALK-rearranged NSCLCs resistant to ALK-TKIs including alectinib, and inhibiting Src signaling could be a potential target for ALK-rearranged NSCLC after ALK-TKI resistance.

Citation Format: Ryohei Yoshida, Shunsuke Okumura, Takaaki Sasaki, Yoshinobu Osaki. Src mediates acquired resistance to ALK inhibitor in ALK-rearranged non-small cell lung cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2121.
Abstract 2122: Downregulation of RGS10 induces PI3K/mTOR inhibitor resistance by Ras activation in glioblastoma
Authors: Li Xiaolong , Wu Shaofang , Koul Dimpy , Yung. W.K. Alfred
Published: July 2016

Glioblastoma multiforme (GBM) is the most common form of malignant brain cancer in adults. The prognosis of affected patients is poor and median survival is only one year. The essential role of PI3K signaling in GBM proliferation, metabolism and differentiation has been highlighted. The major mechanism for the oncogenic activity of PI3K is by preventing apoptosis through the downstream activation of Akt and mTOR. The importance of mTOR in the regulation of cell survival downstream of PI3K signaling provides a strong rationale for the use of dual PI3K/mTOR inhibitor. However, the limited effect of dual PI3K/mTOR inhibitor used as single agent is most likely due to activation of collateral pathways, suggesting that additional refinement of this overall approach is necessary. We investigated potential bypass mechanisms to PI3K/mTOR inhibition as a single agent and generated three resistant cell lines to PI3K/mTOR inhibitor with 3 to 5-fold increase in IC50 value and with an aim to identify the potential bypass of survival to PI3K/mTOR inhibitor. We show that the IC50 of the resistant cell lines and parental lines is correlated with s6 phosphorylation and the three resistant cell lines express increased level of phosphorylated ERK1/2, p70S6K and p90RSK protein levels. Further investigation show that RGS10, a regulator of G protein signaling, is down regulated in the resistant cell lines. Down regulation of RGS10 increases the GTP-bound Ras, implying that Ras activation defines the acquired resistance by activating the ERK/p90RSK pathway. Knockdown RGS10 in the parental cells induces ERK1/2, p70S6K and p90RSK phosphorylation and the resistance to PI3K/mTOR inhibitor. Raf1 or p90RSK knockdown can re-sensitize resistant cell lines to PI3K/mTOR inhibition. Combination of PI3K/mTOR inhibitor with Raf inhibitor Sorafinib efficiently inhibited the growth of the resistant cell lines showing Ras/Raf activation serves as an escape mechanism to PI3K/mTOR inhibition.

These observations suggest that Ras/Raf signaling serves as an survival signal upon PI3K/mTOR inhibition and provides a strong rationale for the combined use of PI3K/mTOR and Raf1 inhibition to induce lethal growth inhibition in human GBM.

Citation Format: Xiaolong Li, Shaofang Wu, Dimpy Koul, W.K. Alfred Yung. Downregulation of RGS10 induces PI3K/mTOR inhibitor resistance by Ras activation in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2122.
Abstract 2126: Androgen receptor expression is associated with sunitinib resistance in renal cell carcinoma models
Authors: Adelaiye-Ogala Remi M. , Chintala Sreenivasulu , Orillion Ashley , Pettazzoni Piergiorgio , Elbanna May , Elzey Bennett , Miles Kiersten Marie , Kao Chinghai , Draetta Giulio F. , Pili Roberto
Published: July 2016

Background: Androgen receptor (AR) expression has been reported in renal cell carcinoma but its biological role remains elusive. Sunitinib is a potent anti-angiogenic drug approved for the treatment of advanced renal cell carcinoma (RCC). However, eventually RCC tumors develop drug resistance. We have hypothesized that tumor cells resistance to sunitinib is associated with kinome reprograming and anti-apoptotic genes upregulation. To date, there is no report on the association between AR expression and resistance to TKI such as sunitinib in RCC. Our study was designed to investigate the role of AR in sunitinib resistance in RCC. We used our previously reported sunitinib resistant ccRCC cells and patient derived xenograft models. Methods: Human RCC cell lines; 786-0, 786-0R (sunitinib resistant), C2, C2R (sunitinib resistant), ACHN and Caki 2 were utilized to determine sensitivity or resistance to sunitinib. Patient derived xenograft (PDX) models of advance and metastatic RCC and RCC cell lines, sensitive and resistant tumors were used to detect AR expression by qRT-PCR, immunohistochemistry and Western blot analysis. Reverse phase protein array (RPPA) was used to assess 249 protein including AR in sunitinib sensitive and resistant tumors. Results: Our qRT-PCR data showed an increase by 1000 folds in mRNA levels of AR in our sunitinib resistant cell lines. Similarly, RPPA data revealed AR to be increased in sunitinib resistant RCC PDX tumors. This observation was confirmed by Western blot analysis. Conclusion: Overall our data suggest the potential role of AR and its association with resistance to sunitinib. Ongoing studies are testing the in vitro and in vivo combination treatment of RCC models with sunitinib and AR antagonists.

Citation Format: Remi M. Adelaiye-Ogala, Sreenivasulu Chintala, Ashley Orillion, Piergiorgio Pettazzoni, May Elbanna, Bennett Elzey, Kiersten Marie Miles, Chinghai Kao, Giulio F. Draetta, Roberto Pili. Androgen receptor expression is associated with sunitinib resistance in renal cell carcinoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2126.
Abstract 2139: RNA nanoparticles as a two-in-one siRNA delivery carrier synergistically suppress drug-resistance cancer via combination therapy with doxorubicin
Authors: Ahn Hyung Jun
Published: July 2016

To overcome multi-drug resistance (MDR) caused by malfunction of genes in chemotherapy, we synthesize RNA nanoparticles (RNA NPs) as a “two-in-one” siRNA delivery system for MDR1 siRNA and BCL2 siRNA by using rolling circle transcription and base-pairing with folate-DNA-cholesterol fragments. The resulting RNA nanoparticles (Dsi RNPs), which contain multiple tandem copies of two siRNA sequences, form the self-assembled structure with about 100 nm of diameter, and simultaneously suppress pump and non-pump phenotypes in the same cancer cells. Using Dsi RNPs, we show that there exists an optimal time point within the window of time at which the synchronous silencing of MDR1 and BCL2 gene markedly enhances the chemosensitivity of MDR cancer cells to doxorubicin. Accordingly, a two-step sequential treatment of MDR cancer cells with Dsi RNPs and doxorubicin leads to the optimal synergistic cancer suppression. Finally, we demonstrate that such a synergism is attributed to the synchronous silencing of both genes, as well as to the precisely controlled timings of drugs and Dsi RNPs treatment. RNA NPs exhibit the folate receptor-specific cellular uptake, lowering the risk of severe adverse side effects. Also, RNA NPs do not induce innate immunostimulatory response and their highly condensed structure reveals the enhanced stability against nuclease attack in bloodstream. Therefore, RNA NPs delivery system provides an alternative tool as a two-in-one siRNA carrier that is suitable for the spatial-temporal synchronization of double gene silencing, and also, Dsi RNPs have great potential as combination chemotherapeutics with traditional anticancer drugs for suppression of MDR cancer.

Citation Format: Hyung Jun Ahn. RNA nanoparticles as a two-in-one siRNA delivery carrier synergistically suppress drug-resistance cancer via combination therapy with doxorubicin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2139.
Abstract 2149: The anti-EGFR antibody mixture Sym004 overcomes acquired resistance to cetuximab in colorectal cancer
Authors: Sanchez-Martin Francisco J. , Dalmases Alba , Bellosillo Beatriz , Argiles Guillem , Gelabert Mariona , Cañadas Israel , Vidal Joana , Siravegna Giulia , Arena Sabrina , Koefoed Klaus , Visa Laura , Arpí Oriol , Horak Ivan D. , Iglesias Mar , Stroh Christopher , Kragh Michael , Rovira Ana , Albanell Joan , Bardelli Alberto , Tabernero Josep , Montagut Clara
Published: July 2016

The anti-EGFR monoclonal antibodies (MoAbs) cetuximab and panitumumab are used to treat ‘RAS’ wild type metastatic colorectal cancer (MCRC), providing significant clinical benefit in patients. However, all patients ultimately develop disease progression, driven by acquisition of mutations in downstream effectors and in the extracellular domain (ECD) of EGFR, in approximately 25% of the cases. Sym004 is a novel 1:1 mixture of two non-overlapping anti-EGFR MoAbs that has recently shown promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored an EGFR mutation in the post-cetuximab tumor sample. Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring EGFR ECD mutations maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As a proof of principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. These data suggest that Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. Overall, EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials.

Citation Format: Francisco J. Sanchez-Martin, Alba Dalmases, Beatriz Bellosillo, Guillem Argiles, Mariona Gelabert, Israel Cañadas, Joana Vidal, Giulia Siravegna, Sabrina Arena, Klaus Koefoed, Laura Visa, Oriol Arpí, Ivan D. Horak, Mar Iglesias, Christopher Stroh, Michael Kragh, Ana Rovira, Joan Albanell, Alberto Bardelli, Josep Tabernero, Clara Montagut. The anti-EGFR antibody mixture Sym004 overcomes acquired resistance to cetuximab in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2149.