bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5850: Salicylate sensitizes prostate cancer (PrCa) to radiotherapy; activation of AMPK and suppression of lipogenesis
Authors: Tsakiridis Theodoros , Marcinko Katarina , Broadfield Linsday , Villani Linda , Gerdes Carrie , Maharaj Danitra , Farell Tom , Wright James , Steinberg Gregory
Published: July 2017

Radiotherapy (RT) is a key therapeutic modality for PrCa that achieves reasonable disease control rates in localized disease. However, this requires dose-escalated RT, which is associated with increased toxicity to surrounding tissues and leads to deterioration of patient quality of life. Improving the therapeutic ratio in PrCa RT is of high clinical importance. Aspirin has an established role in cancer prevention and studies suggested association with improved PrCa outcomes. It was shown that its metabolite salicylate (SAL) binds directly and activates the metabolic stress sensor AMP-activated kinase (AMPK). This is likely achieved through direct binding to the AMPK β1 subunit, leading to enhanced AMPK activity through suppression of AMPK α-subunit T172 de-phosphorylation. In this study we examined the ability of SAL to sensitize PrCa to RT. Hormone insensitive (PC3) and sensitive (LnCap) human PrCa cells, as well as PC3 xenografts grown in immunocompromised nude mice were subjected to SAL and RT treatments. SAL mediated a dose-dependent inhibition of proliferation and clonogenic survival in both PrCa cell lines and suppressed de novo lipogenesis (DNL). This was detected at μM concentrations of the drug, which can be safely achieved in human circulation. SAL (100-500 μM) radio-sensitized PC3 and LnCap cells, and enhanced the ability of SAL to inhibit DNL. In animals treated with conformal RT (10Gy) to the tumor xenografts, Salsalate feeding (chow diet containing 2.5gr/kg) enhanced the cytotoxicity of RT detected as reduced tumor growth kinetics and weight and volume at sacrifice. Salicylate treatment of cells or salsalate feeding of mice led to increased AMPK activity in cells and tumors, inhibitory phosphorylation of Acetyl-CoA Carboxylase and reduction of AktThr308 phosphorylation. These results indicate that SAL could improve radiation response in PrCa through a mechanism involving the suppression of DNL. This is achieved at clinically attainable doses of SAL. This work supports prospective investigation of SAL as a radio-sensitizer in early phase clinical trials of localized PrCa.

Note: This abstract was not presented at the meeting.

Citation Format: Theodoros Tsakiridis, Katarina Marcinko, Linsday Broadfield, Linda Villani, Carrie Gerdes, Danitra Maharaj, Tom Farell, James Wright, Gregory Steinberg. Salicylate sensitizes prostate cancer (PrCa) to radiotherapy; activation of AMPK and suppression of lipogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5850. doi:10.1158/1538-7445.AM2017-5850
Abstract 5852: DUOX2, a key player for chemopotentiation by low-dose fractionated radiation therapy in gastric cancer cells
Authors: Parekh Palak R. , Chang Elizabeth , Sharma Navesh K. , Carrier France
Published: July 2017

One of the most conventional therapy for solid tumors is radiotherapy. Still, this modality presents a challenge when it comes to managing highly disseminated gastrointestinal cancers due to increased toxicity to surrounding tissues. Recent laboratory and clinical data indicate that Low Dose Fractionated Radiation Therapy (LDFRT) can potentiate systemic chemotherapy and presents the possibility to revisit the concept of Whole Abdominal Radiotherapy (WART) for disseminated intra-abdominal gastric cancers. Earlier work considered LDFRT ineffective for tumor removal but we now know that LDFRT induces hyper-radiosensitivity (HRS) in a number of proliferating cells. We have recently shown that dual oxidase (DUOX2) is a major contributor to induce HRS at radiation doses as low as 0.15 Gy and sensitize human gastric cancer cells to chemotherapy. The aim of our study is to determine the utility of DUOX2 as a potential biomarker for the clinical application of chemopotentiation by LDFRT. First, we performed immunohistochemistry (IHC) on 48 human gastric samples with progressive grades. Our data indicate that only about 50% of human gastric cancers are positive for DUOX2. The reason for DUOX2 variability of expression is not clear but may be linked to inflammation since six of the seven (86%) gastritis samples we examined expressed strong levels of DUOX2 in the surface of epithelial cells. Our data also indicate that expression of DUOX2 significantly increases the levels of macrophages infiltration in tissue expressing DUOX2 as well as in the stroma surrounded by cells expressing DUOX2. This suggests that expression of DUOX2 could impact on the dynamic of the tumor microenvironment. Expression of DUOX2 in response to LDFRT is conserved since we also observed this in mice primary gastric cancer cells as well as cancer stem cells. Furthermore, as a first step to develop DUOX2 as an accessible biomarker, we studied DUOX2 activity by measuring the accumulation of oxidative serum proteins in gastric cancer cells media. Our data indicate that down regulation of DUOX2 significantly reduces the levels of serum protein oxidation. Taken together these data suggest that DUOX2 could potentially be used as a biomarker to stratify patients and follow the efficiency of clinical application of chemopotentiation by LDFRT.

Citation Format: Palak R. Parekh, Elizabeth Chang, Navesh K. Sharma, France Carrier. DUOX2, a key player for chemopotentiation by low-dose fractionated radiation therapy in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5852. doi:10.1158/1538-7445.AM2017-5852
Abstract 5853: Peroxiredoxin I-targeted AMRI-59 has a role of radiosensitizer by promoting induction of ROS/γH2AX/caspase pathway and suppression of ERK
Authors: Hong Wan Gi , Cho Jeong Hyun , Kim Ju Yeon , Lee EunAh , Hwang Sang-Gu , Chang Tong-Shin , Um Hong-Duck , Park Jong Kuk
Published: July 2017

We have identified AMRI-59, one of small molecule, is specific pharmaceutical inhibitor of peroxiredoxin (PRX) I enzyme activity in our previous study. In this study, we tried to prove whether AMRI-59 has role of radiosensitizer against non-small cell lung cancer cells - NCI-H460 and NCI-H1299. The radiosensitizer effects of AMRI-59 were tested with clonogenic assays. The intracellular mechanism underlying this effect was determined by performing immunoblotting and measuring ROS generation, mitochondrial potential and cell death. The radiosensitizer activity of AMRI-59 in vivo was tested in nude mice by treating with AMRI-59 and IR, and measuring tumor volume and assessing apoptosis. Dose-enhancement ratios of 30 μM AMRI-59 in NCI-H460 and NCI-H1299 are 1.57 or 3.38, respectively. Combination with AMRI-59 and IR also increased the production of ROS and disruption of mitochondrial potential via enhancement of PrxI oxidation, which subsequently induced one of DNA damage markers - phospho-γH2AX, and suppressed phosphorylation of ERK, and, finally, activated caspase-3. Notably, inhibition of ROS production prevented ERK suppression, and inhibition of ERK in addition of combination with AMRI-59 and IR increased caspase activation and apoptosis. In a xenograft assay, combination with AMRI-59 and IR delayed tumor growth by 11.4 days compared with controls, yielding an enhancement factor of 1.48. Collectively, these results indicate that AMRI-59 functions as a Prx I - targeted radiosensitizer by induction of apoptosis through activation of a ROS/γH2AX/caspase pathway and suppression of ERK.

Citation Format: Wan Gi Hong, Jeong Hyun Cho, Ju Yeon Kim, EunAh Lee, Sang-Gu Hwang, Tong-Shin Chang, Hong-Duck Um, Jong Kuk Park. Peroxiredoxin I-targeted AMRI-59 has a role of radiosensitizer by promoting induction of ROS/γH2AX/caspase pathway and suppression of ERK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5853. doi:10.1158/1538-7445.AM2017-5853
Abstract 5863: Identifying the radiosensitizing effects of PARP inhibitor in ovarian cancer
Authors: Bi Yue , Verginadis Ioannis , Dey Souvik , Guo linlang , Lin Lilie , Zheng Yanfang , Koumenis Constantinos
Published: July 2017

Ovarian cancer is the seventh-most common cancer with a high mortality rate among women. Radiation therapy (RT) is used in ovarian cancer for local relapse, but doses are limited due to concern for small bowel obstruction in patients who have had multiple surgeries and chemotherapeutic regimens. Therefore, it’s an ideal setting to improve the therapeutic window by combining RT and a radiosensitizer. Poly (ADP-ribose) polymerase-1 (PARP-1) is involved in the recognition of DNA damage and the facilitation of DNA repair by recognizing DNA single-strand breaks (SSB). Inhibition of PARP1, especially in tumors driven by BRCA1 or 2 mutations, has been shown to potentiate the DNA-damaging effects of radiation in vitro and in vivo and to increase tumor vasculature perfusion and oxygenation. Approximately half of ovarian cancers have germline or somatic BRCA gene mutation, epigenetic silencing or other mutations that affect homologous recombination (HR) competency resulting in DNA repair defects, making this tumor particularly susceptible to the radiation-sensitizing effects of PARP inhibitors. Here, we investigated the therapeutic effects of the PARP inhibitor olaparib in preclinical models of ovarian cancer. Basic BRCA1 and PARP1 protein and mRNA levels were characterized in a panel of ovarian cancer cell lines. The radiosensitizing effects of olaparib were tested on both HR-proficient and HR-deficient ovarian cancer cells by evaluating the colony formation, DNA damage, apoptosis, and PARP1 activity. Survival benefit of olaparib was also examined in xenograft models of ovarian cancer. Our results demonstrate that combination of olaparib with IR decreased colony formation, inhibited DNA damage (γH2AX) repair, and induced more apoptosis compared to olaparib or IR alone. In addition, HR-deficient cells were more sensitive to IR than HR-proficient cells in the presence of olaparib. Furthermore, olaparib inhibited PARP1 activity in vivo, significantly decreased tumor growth and increased overall survival when combined with IR in mice bearing subcutaneous xenografts of HR-deficient OVCAR8 cells while producing a relative modest effect on overall survival of mice bearing xenografts of HR-proficient SKOV3 cells. These results provide a preclinical rationale for improved treatment modalities using olaparib as an effective radiosensitizer in ovarian cancer, particularly in tumors with HR-deficiencies.

Citation Format: Yue Bi, Ioannis Verginadis, Souvik Dey, linlang Guo, Lilie Lin, Yanfang Zheng, Constantinos Koumenis. Identifying the radiosensitizing effects of PARP inhibitor in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5863. doi:10.1158/1538-7445.AM2017-5863
Abstract 5869: Resistance mechanism against trastuzumab in HER2-positive cancer cells and its negation by Src inhibition
Authors: Jin Meihua , Nam Ah Rong , Park Ji Eun , Bang Ju Hee , Bang Yung Jue , Oh Do Youn
Published: July 2017

Background: Trastuzumab in combination with chemotherapy is a standard of care for patients with HER2-positive breast and gastric cancer. Resistance mechanism to trastuzumab, anti-HER2 therapy, includes multiple pathways. Among them, Src is not well known especially in HER2-positive gastric and biliary tract cancers. We investigated the role of Src involved in trastuzumab resistance and explored the potential of Src inhibition as a trastuzumab resistance overcoming strategy.

Methods: Four trastuzumab-resistant (HR) cells (SNU216HR, N87HR, SNU2670HR, SNU2773HR) were established from 2 HER2-amplified gastric cancer cells (SNU216, NCI-N87) and 2 HER2-amplified biliary tract cancer cells (SNU2670, SNU2773). For Src inhibition, bosutinib was used. MTT assay, colony formation assay, cell cycle analysis by FACS Calibur flow cytometer, and cell migration assay were done. Animal experiments were conducted to test anti-tumor effect of bosutinib using SNU2670 and SNU2670HR xenograft models.

Results: SNU2670HR/NCI-N87HR cells showed pSrc activation, in contrast, SNU216HR/SNU2773HR cells exhibited decreased pSrc expression. In these pSrc decreased HR cells, pFAK was elevated. Bosutinib downregulated Src-FAK signaling more obviously in Src activated HR cells than parental cells. In pSrc decreased HR cells, bosutinib reduced Src-dependent FAK phosphorylation to affect cell fate. Bosutinib inhibited the growth of both parental cells and HR cells, and induced apoptosis and G1 arrest in HR cells. Bosutinib suppressed HR cell migration more effectively compared with parental cells. Bosutinib exhibited potent tumor growth inhibition in both SNU2670 and SNU2670HR xenograft models and more significantly suppressed tumor growth in HR models.

Conclusion: Src activation may contribute to trastuzumab resistance in part in HER2-positive gastric and biliary tract cancer cells. Targeting Src might be a candidate strategy to overcome trastuzumab resistance in HER2-positive cancers.

Citation Format: Meihua Jin, Ah Rong Nam, Ji Eun Park, Ju Hee Bang, Yung Jue Bang, Do Youn Oh. Resistance mechanism against trastuzumab in HER2-positive cancer cells and its negation by Src inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5869. doi:10.1158/1538-7445.AM2017-5869
Abstract 5872: Stat3 feedback activation-induced PTGIS expression is associated with acquired resistance to EGFR-TKI in lung cancer
Authors: Yeh Hsuan-Heng , Wu Shang-Yin , Hung Chun-Hua , Su Wu-Chou
Published: July 2017

Targeted cancer therapies can effectively promote tumor regression in clinical responses. Eventually, most tumors develop resistance to these drugs. Stat3 activation has been suggested as one of the mechanisms that cause acquired resistance to EGFR-tyrosin kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC). However, the underlying molecular mechanisms are not well studied. In this study, we found that treatment of gefitinib (EGFR-TKI) in NSCLC cell lines harboring EGFR-TKI sensitive mutation induced feedback activation of Stat3 in a time- and dose-dependent manner. High levels of IL-6 were detected in the conditioned medium of gefitinib-treated cells by cytokine array and were confirmed by ELISA. We demonstrated that IL-6 treatment could induce Stat3 activation and conditioned medium treatment-induced Stat3 activation was suppressed by IL-6 neutralizing antibody. We also demonstrated that gefitinib treatment-induced IL-6 secretion could be inhibited by knockdown of Stat3 expression. Moreover, pharmacological inhibition and genetic inhibition of Stat3 activation increased cell cytotoxicity of gefitinib in both PC9 and HCC827 cells that harboring EGFR-TKI sensitive mutation. Our data suggested that gefitinib treatment could induce activation of IL-6/Stat3 signaling loop and modulate cell cytotoxicity of gefitinib. Microarray and Ingenuity Pathway Analysis (IPA) analysis revealed prostaglandin I2 synthase (PTGIS) as a downstream target gene of IL-6/Stat3 signaling and PTGIS was up-regulated by gefitinib treatment. The induction of PTGIS by gefitinib via Stat3 feedback activation was confirmed in vitro genetically and pharmacologically. Moreover, we showed that cotreatment with PTGIS inhibitor improves the efficacy of EGFR inhibition in PC9 and HCC827 cells. Importantly, high expression of PTGIS was found in gefitinib-resistant PC9 cells (PC9/gef) compared with gefitinib-sensitive PC9 cells. Targeting PTGIS was also effective in decreasing viability of cells with acquired resistance to gefitinib in PC9/gef cells. Taken together, our study indicated that Stat3 feedback activation-induced PTGIS expression participates in modulating gefitinib efficacy and Stat3/PTGIS inhibition could potentially overcome acquired resistance to gefitinib in NSCLC.

Note: This abstract was not presented at the meeting.

Citation Format: Hsuan-Heng Yeh, Shang-Yin Wu, Chun-Hua Hung, Wu-Chou Su. Stat3 feedback activation-induced PTGIS expression is associated with acquired resistance to EGFR-TKI in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5872. doi:10.1158/1538-7445.AM2017-5872
Abstract 5873: Chaetocin sensitizes GBM cells to TRAIL and BH3 mimetics induced apoptosis
Authors: Özyerli Ezgi , Yeşil Zeynep Kahya , Oppermann Udo , Önder Tuğba Bağcı
Published: July 2017

Glioblastoma Multiforme (GBM) is the most common and aggressive brain tumor, which lacks efficient therapy. TRAIL is an antitumor agent that triggers apoptosis selectively on tumor cells, thus can be utilized as therapeutic approach for GBM. However, most GBM cells confer TRAIL resistance and how this resistance is regulated at a molecular level is not completely clear. Epigenetic deregulation has been increasingly recognized as a hallmark of cancer. In this study, we conducted a drug screen against selected chromatin modifiers in U87MG GBM cells and identified Belinostat, Trichostatin A and Chaetocin as potential apoptosis regulators. While Belinostat and Trichostatin A are well-characterized histone deacetylase inhibitors in the context of malignancies, Chaetocin’s role in cancers is less well-known and its relation to TRAIL is novel. Chaetocin is a fungal mycotoxin that inhibits Suv39H1, a histone methyltransferase (HMT) that catalyzes H3K9 methylation. In this study, we investigated how Chaetocin affects the response of GBM cells to apoptotic stimuli by TRAIL. To this end, GBM cell lines U87MG and U373 are treated with varying dose of Chaetocin and cells’ viability and apoptosis responses to varying doses of TRAIL are analyzed. Chaetocin decreased viability down to 11% of control in U87MG cells upon TRAIL treatment, without inducing cellular toxicity and apoptosis by itself. Chaetocin was shown to upregulate the expression of proapoptotic genes while downregulating antiapoptotic gene expression. In addition, elevated Caspase3/7, Caspase8 and Caspase9 activities were detected in U87MG upon Chaetocin treatment together with PARP cleavage as indicator of apoptosis. Chaetocin also sensitized GBM cells to BH3 mimetic ABT263 which indicates that sensitization of GBM cells by Chaetocin is not limited to TRAIL but is rather a general phenomenon for apoptosis. We infer from these findings that Chaetocin plays critical and specific role in apoptotic pathway. Our future work is directed towards examining the changes in gene expression profiling of GBM cells upon Chaetocin treatment and understanding the functional role of H3K9 Methylation in GBM cell apoptosis. To this end, RNAseq analysis is currently being performed and expected to enlighten Chaetocin’s mechanism of action. These findings support our motivation that identifying epigenetic modifier drugs as apoptosis sensitizer may be a promising therapeutic approach for GBM treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Ezgi Özyerli, Zeynep Kahya Yeşil, Udo Oppermann, Tuğba Bağcı Önder. Chaetocin sensitizes GBM cells to TRAIL and BH3 mimetics induced apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5873. doi:10.1158/1538-7445.AM2017-5873
Abstract 5875: Schlafen 11 (SLFN11) induces lethal S-phase arrest in response to DNA damage: A novel mechanism of how cancer cells are killed by DNA damaging agents
Authors: Murai Junko , Pommier Yves
Published: July 2017

The putative DNA/RNA helicase, Schlafen 11 (SLFN11), is a recently discovered determinant of sensitivity to DNA damaging agents such as platinums, topoisomerase I and II inhibitors (camptothecins, etoposide, doxorubicin), cisplatin, gemcitabine (1-3) and PARP inhibitors (olaparib, talazoparib) (4). Because SLFN11 expression is suppressed in ~45% cancer cell lines, SLFN11 inactivation is potentially one of the most prevalent mechanisms of resistance to DNA damaging agents. However, the molecular mechanisms of SLFN11 action in the DNA damage response have not been solved. Using isogenic cell lines of SLFN11-knockout and SLFN11-overexpression, we will show that SLFN11 binds to chromatin in response to camptothecin within 2 hours. ChIP-seq analysis reveals that SLFN11 preferentially binds to replication origins under camptothecin treatment. We identify two helicases DHX9 and MCM3, which are involved in replication and origin firing, as binding partners of SLFN11 by IP-MS analysis. Cell cycle analyses revealed that SLFN11 inhibits replication in response to camptothecin, which irreversibly lasts until apoptosis is exerted. Notably, the S-phase arrest by SLFN11 is independent of ATR-mediated S-phase checkpoint. Unscheduled origin firing by ATR inhibition under camptothecin treatment is suppressed in the presence of SLFN11. Hence, SLFN11 enforces irreversible S-phase arrest in damaged cells possibly by inhibiting dormant origin firing and inactivating the MCM helicase in conjunction with DXH9. We conclude that the irreversible S-phase arrest by SLFN11 leads to cell death in response to DNA damaging agents, which is a novel mechanism of how cancer cells are killed by DNA damaging agents.

1.

Sousa FG, Matuo R, Tang SW, Rajapakse VN, Luna A, Sander C, et al. Alterations of DNA repair genes in the NCI-60 cell lines and their predictive value for anticancer drug activity. DNA Repair (Amst) 2015;28:107-15.

2.

Zoppoli G, Regairaz M, Leo E, Reinhold WC, Varma S, Ballestrero A, et al. Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents. Proc Natl Acad Sci U S A 2012;109(37):15030-5.

3.

Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 2012;483(7391):603-307.

4.

Murai J, Feng Y, Yu GK, Ru Y, Tang SW, Shen Y, et al. Resistance to PARP inhibitors by SLFN11 inactivation can be overcome by ATR inhibition. Oncotarget 2016.

Citation Format: Junko Murai, Yves Pommier. Schlafen 11 (SLFN11) induces lethal S-phase arrest in response to DNA damage: A novel mechanism of how cancer cells are killed by DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5875. doi:10.1158/1538-7445.AM2017-5875
Abstract 5879: Establishing doxorubicin resistant HCC1806 triple negative breast cancer cell lines result in the differential expression of let7a and miR34a
Authors: Rorie Checo J. , Moses Malcolm M. , Musa Mohammed K. , Godfrey Sherette S.
Published: July 2017

Resistance to chemotherapy is one of the major difficulties in the treatment of triple negative breast cancer. Besides the typical known causes of drug resistance, tumor microenvironment can also promote resistance by preventing the drug from accumulating in tumor cells in order to elicit a cytotoxic response. TNBC is characterized by the loss or low expression of estrogen receptor, progesterone receptor, and HER2 proteins that have been targeted and established as somewhat effective therapy regiments in other breast cancer subtypes including liminal breast cancer. Since TNBC tumors are missing these well-known drug targets, there is little to no advancement in the establishment of drug therapies to accurately target this breast cancer subtype. TNBC is termed as aggressive due to the fact of its invasiveness and metastatic properties. One potential mediator of TNBC aggressive behavior may be due to the fact that this breast cancer subtype may have as high as an 80% p53 tumor suppressor gene mutation rate which may also play a role in the ability of TNBC to recur as a result of the inability to kill all remaining TNBC cells leading to chemotherapy resistance. In these studies, we hope to mimic TNBC recurrence by establishing doxorubicin resistant HCC1806 TNBC cell lines. Here we show that we were able to sustain TNBC cell growth after pulse treatments with increasing concentrations of doxorubicin, and we revealed that p53-related miRNAs, let7a and miR34a, were differentially expressed after pulse treating the HCC1806 cells. Understanding the molecular and mechanistic factors related to recurrence and doxorubicin-resistance in HCC1806 cells may help to develop a list of possible targets for molecular therapy to overwhelm tumor drug resistance in triple negative breast cancer and may develop or establish a list of potential targets for disabling, or combating resistance to doxorubicin treatment in TNBC patients.

Note: This abstract was not presented at the meeting.

Citation Format: Checo J. Rorie, Malcolm M. Moses, Mohammed K. Musa, Sherette S. Godfrey. Establishing doxorubicin resistant HCC1806 triple negative breast cancer cell lines result in the differential expression of let7a and miR34a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5879. doi:10.1158/1538-7445.AM2017-5879
Abstract 5882: FAK is required for the survival of tumor cells in MMTV-Wnt1 driven basal-like mammary tumors
Authors: Paul Ritama , Yeo Syn K. , Luo Ming , Waltz Susan , Guan Jun-Lin
Published: July 2017

Breast cancer is a heterogeneous disease. Stratification of patients based on the subtype of breast cancer is a key to successful treatment of breast cancer. Focal Adhesion Kinase (FAK), a cytoplasmic tyrosine kinase is over expressed and activated in several cancers including breast cancer. Our earlier studies have shown inhibition of FAK in MMTV-PyMT mouse mammary tumors that are classified as Luminal B subtype, delays tumor onset and reduces tumor growth. To address whether inhibition of FAK would be beneficial in basal-like mammary tumors, we generated a conditional deletion of FAK and a knock in mutation of FAK lacking its kinase activity, in MMTV-WNT1 mouse model, which classifies as basal-like. Similar to PyMT mammary tumors we found that loss of FAK or its kinase function delays tumor onset and tumor growth of basal like WNT1 mammary tumors. However unlike the PyMT tumors, the reduced tumor growth in WNT1 model is not due to decreased proliferation. Interestingly loss of FAK activity in WNT1 tumors results in accumulation of cleaved caspase 3, suggesting that loss of FAK activity results in compromised tumor cell survival. When we investigated the pathways through which FAK could affect survival, we found that loss of FAK activity reduces activation of AKT. Reduced AKT activation induces the expression of pro-apoptotic genes. In summary our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells. Hence inhibition of FAK could be beneficial for elimination of basal-like tumor cells.

Citation Format: Ritama Paul, Syn K. Yeo, Ming Luo, Susan Waltz, Jun-Lin Guan. FAK is required for the survival of tumor cells in MMTV-Wnt1 driven basal-like mammary tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5882. doi:10.1158/1538-7445.AM2017-5882
Abstract 5883: CHD1 as regulator of cell adhesion in Ets fusion negative prostate cancer
Authors: Kareddula Aparna , Petrosky Whitney , Tereshchenko Irina , Medina Daniel , Aviv Hana , Singer Eric , DiPaola Robert S. , Hirshfield Kim M.
Published: July 2017

Prostate cancer (PCa) develops into lethal disease in about 10% of men diagnosed with this malignancy.

Common genomic changes are seen in PCa such as alterations in CDKN1B, RB1, TP53, PTEN, NKX3-1, MYC and androgen receptor.

Oncogenic fusions involving the Ets family are seen in 50-70% PCa patients, the most common of which is TMPRSS2-ERG. TMPRSS2-ERG influences cell migration, particularly when PTEN is co-deleted or PIK3CA activating mutations are present. However, in the absence of ERG fusions, molecular drivers of the PCa phenotype are less clear but include alterations in SPOP (6-15% of PCa), SPINK1 (10%), MAP3K7 (18-38%) and CHD1 (15-27%).

CHD1 (Chromo-domain Helicase DNA Binding Protein-1) is a chromatin remodeling factor with many roles including homologous recombination mediated DNA repair of double strand breaks and maintenance of genomic stability and therefore it is hypothesized to cause the predominant intrachromosomal rearrangements observed in Ets fusion negative tumors. This study was undertaken to evaluate the cellular effects that follow CHD1 deletion that could aid PCa initiation and progression. CHD1 was knocked out in the non tumorigenic, HPV16 immortalised prostate epithelial cell line, RWPE-1, using CRISPR/Cas9. Compared to parental RWPE-1 and non target cells (NT2), the CHD1 KO cells had a smaller and rounder morphology and were less adherent to tissue culture plates. To study expression of extracellular matrix and adhesion molecules, RT profiler assay was performed using RWPE-1 parental and CHD1 KO cells. CHD1 KO cells showed a decrease in the expression of SPARC, MMP2, ITGA2, ITGA5, ITGA6, FN1, LAMB3, collagen,

tenascin and vitronectin as compared to parentals and NT2. Reduction in extracellular matrix and adhesion molecules and altered morphology of CHD1 KO cells as compared to parentals and NT2 cells suggest that loss of CHD1 reduces cell-cell and cell-matrix adhesion.

These results suggest that in the absence of PTEN loss in Ets negative tumors, deletion of CHD1 gene could change cell adhesion dynamics ascribing a new role for CHD1 in the development and progression of PCa.

Citation Format: Aparna Kareddula, Whitney Petrosky, Irina Tereshchenko, Daniel Medina, Hana Aviv, Eric Singer, Robert S. DiPaola, Kim M. Hirshfield. CHD1 as regulator of cell adhesion in Ets fusion negative prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5883. doi:10.1158/1538-7445.AM2017-5883
Abstract 5887: The role of FAK in tumor microenvironment
Authors: Wu Hsin-Jung , Yeo Syn Kok , Wilson Megan
Published: July 2017

Focal Adhesion Kinase (FAK) is a cytoplasmic non-receptor tyrosine kinase important in mediating signal transduction in cell migration, survival and proliferation. Up-regulation of FAK in the tumor cells has been shown to enhance tumor malignancy and correlate with poor prognosis. FAK signaling has also been implicated in impacting tumor microenvironment, including endothelial cells and fibroblasts. However, much less is known about how FAK in the tumor stroma affects tumor progression, or how stromal FAK cross-talks with cancer cells. Here we use an inducible Col1α2-creERT, FAK f/f genetic engineered mouse model (GEMM) to specifically delete FAK in the fibroblasts. Using the GEMM, we found that FAK in stromal fibroblasts facilitates breast tumor growth and metastasis in vivo. We further showed that conditioned media (CM) from human breast cancer cells induced FAK Y397 autophoslphorylation and expression of smooth muscle actin (SMA, a marker for cancer-associated-fibroblasts [CAFs]) of normal human lung fibroblasts. Interestingly, only the breast cancer CM educated fibroblasts, but not the naive un-treated fibroblasts, were able to promote breast cancer cell migration and invasion. Lastly, knockdown of FAK in these “educated” fibroblasts abolished their ability to promote breast cancer cell migration and invasion. Taken together, these data suggest a potential role of stromal FAK in creating a pro-tumorigenic microenvironment for breast cancer development and progression, and provide further rationale in therapeutic benefits of FAK inhibition against various solid tumors.   

Citation Format: Hsin-Jung Wu, Syn Kok Yeo, Megan Wilson. The role of FAK in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5887. doi:10.1158/1538-7445.AM2017-5887
Abstract 5893: Bioenergetic signature from cocultures of pancreatic tumor cell lines and fibroblasts
Authors: Agulló-Ortuño Mª Teresa , Prieto-García Elena , Díaz-García C. Vanesa , Blas Irene Otero , García-Ruíz Inmaculada , López-Martín José A.
Published: July 2017

Pancreatic ductal adenocarcinoma (PDAC) is one of the tumors with greater invasiveness and metastasis. PDAC is associated with a large desmoplastic reaction, characterized by fibroblastic proliferation and extracellular matrix secretion. Besides, cellular bioenergetics has become a central issue of investigation in cancer biology, because the altered energy metabolism of cancer cells has been proposed as a potential target for cancer treatment. We have established co-cultures between PDAC cell lines (Capan-1 or PL-45) and fibroblasts (LC5). A reverse phase protein microarray (RPMA) approach has been applied to quantify proteins of energy metabolism in theses co-cultures, with the aim of identifying potential biomarkers in PDACs. The fifteen proteins of energy metabolism studied included members of the mitochondrial oxidation of pyruvate, the tricarboxylic acid cycle, β-oxidation of fatty acids, electron transport and oxidative phosphorylation, glycogen metabolism, glycolysis and oxidative stress using highly specific antibodies. Co-cultures modified proteins expression of energy metabolism, respect to monocultures. Capan-1 in co-culture increased several proteins belonging to OXPHOS (NDUFS3, SDHB, CORE2 and COXII), the antioxidant SOD2, the mitochondrial HSP60, and G6PDH. The expression of the glycolitic PKM2 decreased. Their fibroblasts partners increased the expression of SDHB and also decreased PKM2. PL-45 in co-culture, experimented an increase in HADHA. Their fibroblasts partners showed a decrease in PKM2, LDHA, IF1, PDHe and HSP60.Capann-1 and PL-45 in co-culture with fibroblast, differ en their energy metabolism phenotype. Overall, the results indicate that the quantification of bioenergetic signature offers potential biomarkers that could be implemented to refine the understanding of the biological principles of PDAC and, eventually, the management of patients with these tumors.

Citation Format: Mª Teresa Agulló-Ortuño, Elena Prieto-García, C. Vanesa Díaz-García, Irene Otero Blas, Inmaculada García-Ruíz, José A. López-Martín. Bioenergetic signature from cocultures of pancreatic tumor cell lines and fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5893. doi:10.1158/1538-7445.AM2017-5893
Abstract 590: A new standardized CD8 and PD-L1 dual assay
Authors: Monville Florence , Prestat Emmanuel , Yessaad Nadia , Villard Marine , Batista Luciana , Galon Jerome , Adam Julien , Fieschi Jacques
Published: July 2017

PD1/PD-L1 pathway blockade results in a durable clinical response in a fraction of the non-small cell lung cancer (NSCLC) patients. Today, the expression of PD-L1, detected by immunohistochemistry (IHC) at the surface of tumor or tumor-infiltrating immune cells, is used to select patients that may respond to immune checkpoint inhibitors (ICI). However the predictive value of that biomarker alone is questioned. In order to better stratify NSCLC patients, we have developed a new dual-staining IHC assay of PD-L1+ and CD8+ cells (TILs) present in the tumor microenvironment on a single slide prepared from FFPE tissue.

The assay was optimized and fully automated on the Benchmark XT platform (Roche Ventana) with anti-PD-L1 (HDX3) and anti-CD8 (HDX2) monoclonal primary antibodies, respectively revealed with DAB and fast red substrates. Stained slides are analyzed by a pathologist like any other existing IVD test together with the TILs information. In addition, after digitization, the samples are analyzed to quantify brown and red cells with a newly developed digital Pathology (DP) tool.

The following parameters are reported by the DP tool: (1) CD8+ cell density (cells/mm²) ; (2) PD-L1+ cell density (cells/mm²) ; proximity between CD8+ and PD-L1+ cells, either centered on CD8+ or on PD-L1+ cells and finally cluster indexes for CD8+ cells and PD-L1+ cells. HalioDx DP analysis tool can calculate these parameters on any Regions Of Interest (ROI) defined by the user.

Accuracy of the automated numeration of CD8+ and PD-L1+ cells was validated by an expert pathologist. Cell-to-cell distances were validated with an independent DP tool. Variability was assessed for all parameters using adjacent dual-stained slides on complete tissue sections. The concordance with main IVD approved PD-L1 methods was established on a representative set of 55 NSCLC tumours.

HalioDx has developed an innovative assay based on the dual staining of CD8+ and PD-L1+ cells associated to a DP tool to standardize the evaluation of the tumor microenvironment. This assay could have higher predictive performance than existing IVD tests used to identify ICI resistant NSCLC patients.

Citation Format: Florence Monville, Emmanuel Prestat, Nadia Yessaad, Marine Villard, Luciana Batista, Jerome Galon, Julien Adam, Jacques Fieschi. A new standardized CD8 and PD-L1 dual assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 590. doi:10.1158/1538-7445.AM2017-590
Abstract 5905: Cancer associated fibroblasts promote tumor metastasis coexisting with cancer cells in blood circulation
Authors: Kashima Hajime , Noma Kazuhiro , Sato Hiroaki , Katsura Yuki , Kato Takuya , Ohara Toshiaki , Tazawa Hiroshi , Shirakawa Yasuhiro , Fujiwara Toshiyoshi
Published: July 2017

Background: Cancer associated fibroblasts (CAFs) are activated fibroblasts and an important player in the tumor microenvironment. Their activity promotes cancer cell proliferation, migration, and invasion, and metastasis. The most prognostic factor in tumor progression is metastasis. Cancer metastasis is a multi-step process that tumor cells detach from primary site, survive in the bloodstream, and seed in target organ. Although previous studies have focused on the interaction between CAFs and cancer cells in primary tumor site, the roles of CAFs in blood circulation remain largely unknown. We investigated the effect of CAFs coexisting with cancer cells in bloodstream on tumor metastasis in vivo mouse model, and also examined the effect of CAFs in subcutaneous tumor.

Methods: We used female BALB/c-nu/nu mice and BALB/c mice in the experiments. Cancer cells were mouse mammary carcinoma cell lines 4T1 transfected with luciferase, colon cancer cell lines Colon 26 transfected with luciferase. Fibroblast cell lines were mouse embryonic fibroblast MEF and NIH-3T3. In venous injection mouse model, we injected 1 × 106 cancer cells alone or the same number of cancer and fibroblast co-cultured together. When we injected cancer cells transfected with luciferase, the mice were subjected to in vivo imaging system (IVIS) and measured luminescence intensity of metastatic sites. We have also harvested lung and compared its weight and metastatic nodule under microscopy. Furthermore, in subcutaneous tumor metastatic mouse model, cancer cells alone or mixed with cancer cells and CAFs were subcutaneously inoculated into the mice.

Results: In the group of mice injected with cancer cells and fibroblasts, luminescent intensity of each lungs were higher than cancer cell alone. Harvested lung weight and the number of metastatic nodules were also higher in the group with cancer cells and fibroblasts.In subcutaneous model, mice inoculated with cancer cells and fibroblast had much more metastatic sites than cancer cells alone.

Conclusions: Our data indicate that CAFs promote tumor metastasis by stimulating cancer cells in blood circulation, as well as in primary tumor site.These findings suggest that CAFs in both bloodstream and primary site could be a promising therapeutic target. CAF-targeted therapy could reduce tumor metastasis and improve the prognosis of cancer patients.

Citation Format: Hajime Kashima, Kazuhiro Noma, Hiroaki Sato, Yuki Katsura, Takuya Kato, Toshiaki Ohara, Hiroshi Tazawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Cancer associated fibroblasts promote tumor metastasis coexisting with cancer cells in blood circulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5905. doi:10.1158/1538-7445.AM2017-5905
Abstract 5907: Radiotherapy-induced damage to cancer-associated fibroblasts and its reciprocal influence on cancer progression: Impact of soluble factors
Authors: Wever Olivier De , Tommelein Joke , Bracke Marc , Boterberg Tom , Demetter Pieter , Verset Laurin
Published: July 2017

Preoperative radiotherapy is a mainstay in current management of colorectal cancer. Several tumor types, including colorectal cancer, show an abundant desmoplastic stroma, characterized by cancer-associated fibroblasts (CAFs). Nevertheless the effects of radiation to the CAFs in the stroma and its reciprocal impact on the response of CRC cells to irradiation have not been studied. We investigated the functional (cell number, morphotype) and biochemical (transcriptomics, metabolomics and kinomics) phenotype of CRC cells in response to paracrine signals from irradiated CAFs. Using neutralizing antibodies and small molecule inhibitors, we analysed the role of tyrosine-kinase receptor activation in CRC cells in response to supernatants from irradiated CAFs. Furthermore, the impact of tyrosine-kinase receptor neutralization in combination with RT was evaluated in an orthotopic CRC model. The level of mTOR activation, a downstream tyrosine-kinase receptor intermediate, was analysed in response to neoadjuvant treatment in human CRC specimens. Further work will reveal which soluble factor(s) are implicated in the communication between irradiated CAFs and colorectal cancer cells.

Citation Format: Olivier De Wever, Joke Tommelein, Marc Bracke, Tom Boterberg, Pieter Demetter, Laurin Verset. Radiotherapy-induced damage to cancer-associated fibroblasts and its reciprocal influence on cancer progression: Impact of soluble factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5907. doi:10.1158/1538-7445.AM2017-5907
Abstract 5908: Cancer-associated fibroblast driven drug resistance in colorectal cancer
Authors: Garvey Colleen M. , Chen Oscar , Mumenthaler Shannon M.
Published: July 2017

Drug resistance remains a major problem in the treatment of most cancers. For example, KRAS wild-type colorectal cancers (CRCs) are typically treated with the anti-epidermal growth factor receptor (EGFR) therapy cetuximab in combination with standard chemotherapy; however, of the 40% of patients that do respond, virtually all relapse within 3-12 months. Additionally, around 25% of nonresponders have no identifiable resistance mechanisms. This suggests that it is not just cell-intrinsic mechanisms that result in resistance, but that extrinsic factors play a role. The purpose of this study was to investigate how the dynamics of the tumor microenvironment, specifically cancer-associated fibroblasts (CAFs) and hypoxia, modify the response of CRC cells to cetuximab. We used a novel high-content imaging platform to generate quantitative phenotypic data (i.e. morphology, birth/death rates) of cellular co-cultures perturbed by multiple, co-occurring microenvironmental conditions. Preliminary data demonstrated reduced sensitivity of tumor cells to cetuximab in the presence of CAFs, which was dependent on patient specifics and time scale of co-culture. Additionally, we found that low oxygen conditions altered the effect of CAFs on tumor cell phenotypes, which highlights the importance of studying co-occurring microenvironmental factors. This work underlies the importance of considering not just the genetic makeup of patient tumors, but also the heterogeneity of the surrounding microenvironment when designing personalized treatment strategies.

Citation Format: Colleen M. Garvey, Oscar Chen, Shannon M. Mumenthaler. Cancer-associated fibroblast driven drug resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5908. doi:10.1158/1538-7445.AM2017-5908
Abstract 591: Efficacy of ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic tumor models alone and in combination with immune checkpoint inhibitors
Authors: Losey Heather C. , Lopes Jared E. , Dean Reginald L. , Huff Michael R. , Moroso Rosemarie A. , Alvarez Juan C.
Published: July 2017

ALKS 4230 is a novel immunotherapeutic agent being tested in an ongoing phase 1 study to evaluate safety and tolerability in the treatment of patients with refractory solid tumors. ALKS 4230 is a selective agonist of the intermediate-affinity interleukin 2 (IL-2) receptor that when administered to mice results in a significant increase in numbers of memory CD8+ T cells and NK cells with no expansion of CD4+ regulatory T cells (Tregs). The selectivity achieved by ALKS 4230 is due to the fact that the molecule is a fusion of circularly permuted IL-2 to the extracellular portion of the IL-2 receptor α, and the resulting fusion protein is sterically prevented from binding to the high-affinity IL-2 receptor expressed on Tregs. The efficacy of ALKS 4230 was compared to recombinant human IL-2 in a B16F10 lung tumor metastasis model. ALKS 4230 treatment resulted in dose-dependent reduction of lung tumor colonization, with 100% inhibition at the highest dose tested. In contrast, while IL-2 was able to reduce lung tumor colonization, the maximal level of inhibition achieved was 60-70% at multiple dose levels such that increasing doses did not result in greater inhibition. Thus, the activation and expansion of effector cells without expansion of Tregs in response to ALKS 4230 treatment correlates with its improved efficacy over IL-2, which non-selectively expands both effector cells and Tregs. The antitumor efficacy mediated by ALKS 4230 was further evaluated in several murine subcutaneous syngeneic tumor models, including B16F10, MC38 and EMT6. Treatment with ALKS 4230 or its murine ortholog resulted in inhibition of tumor growth and improved survival in multiple models. When dosed in combination with anti-CTLA-4 or anti-PD-1 antibodies, ALKS 4230 resulted in further improvement of tumor growth inhibition and survival. These results demonstrate the murine antitumor efficacy of ALKS 4230 alone and in combination with immune checkpoint inhibitors and support the ongoing clinical evaluation of ALKS 4230 as an immunotherapy for cancer.

Citation Format: Heather C. Losey, Jared E. Lopes, Reginald L. Dean, Michael R. Huff, Rosemarie A. Moroso, Juan C. Alvarez. Efficacy of ALKS 4230, a novel immunotherapeutic agent, in murine syngeneic tumor models alone and in combination with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 591. doi:10.1158/1538-7445.AM2017-591