bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1583: SRI31215, a novel inhibitor of oncogenic HGF/MET signaling
Authors: Owusu Benjamin Y. , Venukadasula Phanindra K. , Galemmo Robert A. , Klampfer Lidija
Published: July 2016

Constitutive activation of MET signaling, frequently observed in cancer, triggers signaling by AKT, ERK 1/2 and STAT3, and promotes survival, proliferation, migration and invasion of cancer cells. Accordingly, MET amplification or overexpression of its ligand, hepatocyte growth factor (HGF), are associated with tumor aggressiveness, resistance to therapy and poor prognosis in many cancer patients. Cancer cells can secrete HGF which triggers autocrine HGF/MET signaling, however, fibroblasts are the predominant source of HGF and activate MET expressed on tumor cells in a paracrine manner. Cancer cells and fibroblasts secrete HGF as an inactive precursor, pro-HGF. A crucial step in the regulation of HGF/MET signaling is the proteolytic processing of the inactive precursor, pro-HGF, to its active form by one of the three serine proteases, matriptase, hepsin or HGF activator (HGFA). This is the rate-limiting step in the HGF/MET signaling pathway, controlled by the inhibitors of HGF activation, hepatocyte growth factor activator inhibitors 1 and 2 (HAI-1/2).

We synthesized a novel small molecule, SRI31215, a triplex inhibitor of matriptase, hepsin and HGFA, mimicking the biological activity of HA1-1/2. We confirmed that SRI31215 inhibits the proteolytic activation of pro-HGF and blocks HGF-induced MET activation and signaling by AKT, ERK 1/2 and STAT3. SRI31215 blocked fibroblast-induced epithelial-to-mesenchymal transition (EMT) and prevented scattering and migration of cancer cells. Finally, we demonstrated that SRI31215 sensitized HGF-producing colon cancer cells to epidermal growth factor receptor inhibitors (EGFRi) and inhibited fibroblast-mediated, HGF-dependent, resistance to EGFRi in colon cancer. Thus, SRI31215 blocks signaling between cancer cells and fibroblasts and inhibits the tumor-promoting activity of cancer associated fibroblasts.

Taken together, our data demonstrate that inhibitors of HGF activation, such as SRI31215, represent a novel approach to curb the growth and metastatic spread of tumor cells that are addicted to HGF/MET signaling and to overcome primary and acquired resistance to EGFR-targeted therapy.

Citation Format: Benjamin Y. Owusu, Phanindra K. Venukadasula, Robert A. Galemmo, Lidija Klampfer. SRI31215, a novel inhibitor of oncogenic HGF/MET signaling. [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 1583.
Abstract 159: Analysis of resistance to the combination of a PI3K- and Parp-inhibitor using a genomic sequencing approach
Authors: Nabavi Sheida , Juvekar Ashish , Wang Nicholas , Cantley Lewis C. , Wulf Gerburg M.
Published: July 2016

The objective of this study is to investigate the genomic and transcriptonal changes that are associated with resistance to treatment with the combination of a PI3K-inhibitor (NVP-BKM120) and a PARP-inhibitor (Olaparib). Previous studies have shown that the combination of a PI3K-inhibitor and a PARP-inhibitor synergistically reduced the growth of BRCA-related xenograft tumors derived from patients with TNBC. However, in clinical practice, primary and secondary resistance to this combination is observed. We hypothesize that acquired resistance is the result of a genomic evolution resulting from the selection pressure exerted by the drug treatment.

We used a genetically engineered mouse model, K14-Cre BRCA1f/fp53f/f, where primary tumors had been propagated in Cre-negative littermates and treated to point of resistance with NVP-BKM120 or NVP-BKM120 plus Olaparib. For each of the three tumors, we analyzed exome sequencing and RNA-seq using Illumina technology at the stage of sensitivity (C), resistance to PI3K-inhibition (B) and resistance to the combination (BO); i.e. for each individual tumor a parental clone and its drug-resistant subclones, obtained in vivo, were analyzed. We used the Mutect software tool to call somatic mutations, VarScan2 to call somatic copy number variations, Cuffdiff for differential expression analysis, and TophatFusion. Data were integrated to identify genes for functional analyses using MetaCore and MSigDB software tools.

We found that tumors resistant to the combination drug treatment in general had fewer gross genomic alterations (CNVs) than parental tumors, indicative of evolution of a less variable subclone. However, these tumors also displayed a higher number of non-synonymous mutations than their parental clone. Pathway analysis showed that somatically mutated genes in PI3K-inhibitor resistant tumors were highly enriched for antigen processing and presentation, while those of tumors resistant to the combination treatment were enriched for histone modification pathways. Notably, the tumors resistant to PI3K-inhibitor alone were enriched for Insulin-processing pathways. Genomic losses in PI3K-inhibitor treated tumors enriched for the estrogen receptor pathway (ESR) in breast cancer and DNA damage pathways.

In conclusion, our experimental design of analyzing isogenic tumors resistant to PI3K- or combined PI3K- and Parp-inhibitors enabled us to identify genomic alterations and pathways that explain the evolution to drug resistance.

Citation Format: Sheida Nabavi, Ashish Juvekar, Nicholas Wang, Lewis C. Cantley, Gerburg M. Wulf. Analysis of resistance to the combination of a PI3K- and Parp-inhibitor using a genomic sequencing approach. [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 159.
Abstract 1598: LncRNA AK001796 as a therapeutic target in aggressive breast cancers
Authors: Kumar Maneesh , Sinnott DeVaux Rebecca , Shen Julia J. , Davis Steven P. , Dinger Marcel E. , Mattick John S. , Perou Charles M. , Rosen Jeffrey M. , Mani Sendurai A. , Herschkowitz Jason I.
Published: July 2016

Breast cancer is a heterogeneous disease that can be classified into several distinct molecular subtypes based on gene expression. Like mRNAs and miRNAs, long noncoding RNAs (lncRNAs) differ dramatically in expression across subtypes and can be used for classification. While there has been considerable emphasis on miRNAs, our knowledge is still lacking about the role of lncRNAs that comprise the majority of the mammalian transcriptome. Recently, the importance of lncRNAs in cancer has been highlighted by several studies. We have examined the expression profiles of >17,000 lncRNAs in a large set of breast tumors and have identified a lncRNA, AK001796, that is overexpressed in aggressive breast cancers. In particular, AK001796 is enriched in the aggressive claudin-low, HER-enriched, and luminal B subtypes. Furthermore, in four different models, we find that AK001796 is significantly upregulated in cell lines induced to undergo EMT and in putative mesenchymal-like cancer stem cells within cell lines suggesting this lncRNA as an inducer/facilitator of EMT. Similar results were obtained when a lung cancer cell line was induced to EMT through TGF beta treatment. Using cell fractionation, we have discovered that AK001796 is maintained predominantly in the nucleus. By RACE we have identified two isoforms of AK001796 in breast cancer cells that differ by the presence or absence of a 94 nucleotide intron. The short form (with intron spliced out) appears to be the variant enriched following EMT and may serve as a marker of aggressiveness. Interestingly, knockdown of AK001796 using antisense oligonucleotides lead to significantly increased apoptosis in EMT positive cell lines whereas in EMT negative cells knockdown had little effect. Preliminary studies for finding out the protein interacting partners identified some mesenchymal phenotype-associated proteins in pull-down studies using biotinylated oligos. To further investigate the molecular mechanisms regulated by AK001796 and its utility as a therapeutic target, we will determine the pathways induced by AK001796 by mapping its protein interaction network and downstream signaling pathways. These results nominate AK001796 as a promising therapeutic target in aggressive breast cancers.

Citation Format: Maneesh Kumar, Rebecca Sinnott DeVaux, Julia J. Shen, Steven P. Davis, Marcel E. Dinger, John S. Mattick, Charles M. Perou, Jeffrey M. Rosen, Sendurai A. Mani, Jason I. Herschkowitz. LncRNA AK001796 as a therapeutic target in aggressive breast 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 1598.
Abstract 1600: Polo-like kinase 1 induces epithelial-to-mesenchymal transition and promotes epithelial cell motility by activating CRAF/ERK signaling
Authors: Wu Jianguo , Ivanov Andrei I. , Fu Zheng
Published: July 2016

Polo-like kinase 1 (PLK1) is a key cell cycle regulator that has been implicated in the development of various cancers, including prostate cancer. However, the functions of PLK1 beyond cell cycle regulation remain poorly characterized. Here, we report that PLK1 overexpression in prostate epithelial cells triggers oncogenic transformation. It also results in dramatic transcriptional reprogramming of the cells, leading to epithelial-to-mesenchymal transition (EMT) and stimulation of cell migration and invasion. Consistently, PLK1 downregulation in metastatic prostate cancer cells enhances epithelial characteristics and inhibits cell motility. The signaling mechanisms underlying the observed cellular effects of PLK1 involve direct PLK1-dependent regulation of CRAF with subsequent stimulation of the MEK1/2-ERK1/2-Fra1-ZEB1/2 signaling pathway. Our findings highlight novel non-canonical functions of PLK1 as a key regulator of EMT and cell motility in normal prostate epithelium and prostate cancer. This study also uncovers a previously unanticipated role of PLK1 as a potent activator of MAPK signaling.

Citation Format: Jianguo Wu, Andrei I. Ivanov, Zheng Fu. Polo-like kinase 1 induces epithelial-to-mesenchymal transition and promotes epithelial cell motility by activating CRAF/ERK signaling. [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 1600.
Abstract 1610: Inhibiting HuR, a RNA-binding protein, for inhibition of pancreatic cancer EMT and CSCs
Authors: Dong Ruochen , Polireddy Kishore , Zhang Ying , Chen Qi
Published: July 2016

Purpose

Epithelial- mesenchymal transition (EMT) contributes importantly to cancer cell metastasis, and formation of cancer stem cells (CSCs). An RNA-binding protein, HuR, plays an important role in many solid tumors for promoting cell proliferation, metastasis, anti-apoptosis and drug resistance. However, the role of HuR in cancer cell EMT and CSC has not been elucidated. Here we aim to investigate the role of HuR in pancreatic cancer EMT and CSC, and developing a new HuR inhibitor ST-3 as an inhibitor for pancreatic cancer EMT and CSCs.

Methods

Fluorescence Polarization assay and surface plasmon resonance assay was utilized for St-3, HuR and target mRNA binding. Scratching assay and matrigel invasion assay were used for cell migration and invasion, whereas MTT assay for viability. Tumor spheroid formation assay was used as an indication of CSCs. Western blot and RT-qPCR was used for protein and mRNA expression.

Results

Knockdown of HuR with siRNA inhibited migration of pancreatic cancer cells MIAPaCa-2, and suppressed the expression of Vimentin, Snail, and increased E-cadherin expression, showing inhibition in EMT. The suppression of Snail is due to accelerated mRNA decay. Overexpression of HuR protected mRNA of Snail from degradation, suggesting that Snail is a direct target of HuR. Knockdown of HuR also decreased pancreatic cancer spheroids formation.

St-3 inhibited the proliferation of pancreatic cancer cell lines more potently in HuR-high cells MIAPaCa-2 (IC50 ∼2 μM), than in HuR-low cells BxPc-3 and PANC-1 (IC50 6-20 μM). St-3 directly bound to HuR, and inhibited binding of HuR with its target mRNAs. St-3 treatment mimicked the HuR knockdown effects in inhibiting cell migration, EMT, and spheroids formation.

Conclusion

HuR is an important regulator in pancreatic cancer EMT and CSCs. ST-3 as a novel HuR inhibitor inhibited pancreatic cancer EMT and CSCs. Further investigation is under way.

Citation Format: Ruochen Dong, Kishore Polireddy, Ying Zhang, Qi Chen. Inhibiting HuR, a RNA-binding protein, for inhibition of pancreatic cancer EMT and CSCs. [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 1610.
Abstract 1616: Galectin-4 interferes with the integrin β4/Src/FAK cascade and attenuates metastasis in urothelial carcinoma
Authors: Lin Li-Fang , Wu Meei-Maan , Lee Te-Chang
Published: July 2016

Galectin-4 (Gal-4), a multifunctional lectin, is presented at both intracellular and extracellular sites. Recent studies have shown that Gal-4 plays certain role in attenuating cancer progression and metastasis. We have also observed that patients with urothelial carcinoma (UC) with the lower Gal-4 expression were at higher grade and stage. In addition, the Gal-4 expression is inversely associated with lymph node invasion and prognosis. Since how Gal-4 attenuates the cancer metastasis in UC has not been well investigated, the goal of the present study is to understand the role of Gal-4 in UC progression. We ectopically expressed Gal-4 in UC cells and found that increased Gal-4 significantly inhibits cell proliferation, colony formation, migration, and invasion. We also demonstrated that Gal-4 overexpression leads to a significant decrease the adhesion of UC cells to the basement membrane substrate laminin. Mechanistically, we found that Gal-4 overexpression significantly reduces the expression of the laminin receptor integrin α6β4. Besides to downregulation of integrin β4, Gal-4 overexpression also reduces phosphorylation of Src and focal adhesion kinase (FAK) and promotes anoikis. Together, the present results revealed that Gal-4 reduces the metastasis activity of UC cells by interfering integrin β4/Src/FAK cascade, which are of essential to prevent the suspended cells from anoikis. These results suggest that Gal-4 could be considered as a pivotal factor in the migration and invasion of UC cells.

Key words: Galectin-4, integrin β4, migration, invasion, metastasis, anoikis, urothelial cancer

Citation Format: Li-Fang Lin, Meei-Maan Wu, Te-Chang Lee. Galectin-4 interferes with the integrin β4/Src/FAK cascade and attenuates metastasis in urothelial 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 1616.
Abstract 1618: Thromboxane A2 receptor controls migration and metastasis of triple-negative breast cancer cells
Authors: Zhang Yuanqin , Zhang Xuejing , Nie Daotai
Published: July 2016

Background: Triple negative breast cancers (TNBCs) have poor prognosis and there are no effective targeted therapies available. Thromboxane A2 receptor (TBXA2R) is a G protein coupled receptor whose ligand is a prostanoid produced by cyclooxygenase and thromboxane A2 synthase. There are two isoforms of TBXA2R as results of alternative splicing. TBXA2Rα and β differ in the cytoplasmic tail distal at amino acid residue 328. High expression of TBXA2R has been linked with poor progression free survival of breast cancer cells, but it is unknown how TBXA2R contributes to the progression of breast cancers and which isoform(s) is involved.

Methods: TBXA2R expression in breast cancer was determined by RT-PCR, Western blot and immunohistochemistry using isoform specific primer sets or antibodies. Small hairpin RNAs and CRISPR-CAS9 were used to knock down or knock out TBXA2R expression. Tumor cell migration was evaluated by wound healing and Boyden chamber assays. Cell proliferation and survival was evaluated by MTS and trypan blue based cell counting. Xenograft models were used to assess breast tumor growth, progression, and metastasis.

Results: While TBXA2Rα is ubiquitously expressed in breast cancer cells, TBXA2Rβ is selectively expressed in triple negative breast cancer. Activation of TBXA2R had no discernible effects on proliferation or survival of MDA-MB-231 cells. However, TBXA2R activation led to rapid activation of RhoA and cytoskeletal reorganizations of TNBC cells. TBXA2R activation also led to rapid activation of protein kinase N1 (PKN1), a kinase frequently amplified in TNBCs. Inhibition or depletion of TBXA2R led to reduced tumor cell migration in vitro and prevented breast cancer metastasis in vivo. Further analyses found that inhibition or depletion of TBXA2R compromised the ability of TNBC cells to extravasate in an animal model.

Conclusions: Our data suggest that TBXA2R can contribute to TNBC progression and metastasis by controlling tumor cell cytoskeleton reorganization and motility.

Citation Format: Yuanqin Zhang, Xuejing Zhang, Daotai Nie. Thromboxane A2 receptor controls migration and metastasis of triple-negative 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 1618.
Abstract 1623: CITED2 modulates metastatic ability through effects on IKK-alpha in breast cancer cells
Authors: Jayaraman Swaathi , Doucet Michele , Lau Wen M. , Kominsky Scott L.
Published: July 2016

Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain-2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators. Previously, we identified CITED2 as a novel facilitator of bone metastasis in murine mammary cancer. Extending these initial studies to human breast cancer, CITED2 mRNA expression was significantly elevated in patient samples of metastatic breast cancer relative to primary tumors, with highest levels in bone metastasis. To explore the functional impact of CITED2 in breast cancer metastasis, we stably reduced CITED2 expression in the human breast cancer cell lines MDA-MB-231 and MDA-MB-468 cells, which are metastatic in animal models. While CITED2 knockdown had no effect on cell proliferation, it significantly reduced tumor migration and invasion in vitro and metastasis following intra-cardiac administration in athymic nude mice in vivo. To explore the mechanism responsible for these effects, we examined the impact of CITED2 knockdown on gene expression in MDA-MB-231 cells by microarray analysis. As confirmed at the mRNA and protein levels in both MDA-MB-231 and MDA-MB-468 cells, expression of the NF-κB regulator IKKα was significantly reduced along with several downstream targets of the NF-κB pathway (OPN, MMP9, uPA, SPARC, IL11 and IL-1β). In line with the reduced expression of IKKα, both canonical and non-canonical NF-κB signaling was inhibited following CITED2 knockdown. By ChIP assay, CITED2 was recruited to the promoters of IKKα, as well as the NF-κB targets OPN, MMP9, and SPARC. Finally, restoration of IKKα expression in MDA-MB-231 and MDA-MB-468 cells following CITED2 knockdown rescued their invasive ability. Collectively, these results suggest that CITED2 exerts its pro-metastatic effects in breast cancer cells via regulating IKKα.

Citation Format: Swaathi Jayaraman, Michele Doucet, Wen M. Lau, Scott L. Kominsky. CITED2 modulates metastatic ability through effects on IKK-alpha 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 1623.
Abstract 1627: Kinase-independent function of focal adhesion kinase in lung metastasis of breast cancer
Authors: Luo Ming , Wicha Max S. , Guan Jun-Lin
Published: July 2016

Focal adhesion kinase (FAK) has been shown to promote mammary tumorigenesis, lung metastasis and the maintenance of cancer stem cells in the MMTV-PyMT breast cancer model. Using mammary epithelial cell (MaEC)-specific FAK knockout and kinase-defective (KD) knockin mouse models, we recently demonstrated that loss of FAK kinase activity in MaECs specifically impaired luminal progenitor proliferation and alveologenesis, whereas FAK kinase-independent function was sufficient to support ductal invasion and basal mammary stem cell activity. To further elucidate this kinase-independent role of FAK in mammary tumorigenesis and lung metastasis, we established and analyzed three cohorts of PyMT mice expressing wild type FAK (Ctl-MT), KD-FAK (cKD-MT) and FAK-null (cKO-MT) in MaECs. This study revealed that both FAK deletion and KD knockin mutation in MaECs significantly suppressed mammary tumorigenesis and the content of stem-like tumor cells (CD24+CD29+CD61+). However, in contrast to cKO-MT mice that displayed diminished metastatic nodule formation in the lungs, loss of FAK kinase activity by KD knockin mutation did not affect lung metastatic nodule formation. Using a Boyden chamber assay, we further demonstrated that, unlike cKO-MT tumor cells that displayed diminished cell migration induced by fibronectin or TGFβ1 (as compared to Ctl-MT tumor cells), cKD-MT tumor cells exhibited strong fibronectin-, but not TGFβ1-induced migratory activity. Immunofluorescence of primary tumor sections also revealed that both Ctl-MT and cKD-MT tumors displayed strong ERK activation at tumor periphery, whereas cKO-MT tumors had weak ERK activation in this region. Lastly, when treated with or without paclitaxel in culture, we found that paclitaxel was able to induce strong expression of cleaved caspase 3 and p21 in cKO-MT but not Ctl-MT and cKD-MT tumor cells, suggesting a role for FAK kinase-independent function in the suppression of p21 expression and protection of apoptosis. Together, this study strongly implicates a role for FAK kinase-independent function in promoting lung metastasis.

Citation Format: Ming Luo, Max S. Wicha, Jun-Lin Guan. Kinase-independent function of focal adhesion kinase in lung metastasis of 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 1627.
Abstract 163: Genetic difference between multicentric carcinogenesis and intrahepatic metastasis of hepatocellular carcinoma
Authors: Midorikawa Yutaka , Yamamoto Shogo , Tatsuno Kenji , Ueda Hiroki , Tsuji Shingo , Nagae Genta , Takayama Tadatoshi , Aburatani Hiroyuki
Published: July 2016

Multiple hepatocellular carcinoma (HCC) is categorized into 2 types; multicentric hepatocarcinogenesis (MC) and intrahepatic metastasis (IM). Although discrimination of the types of multiple HCC is of clinical importance, it is quite difficult to determine it correctly even after histological diagnosis. In order to classify multiple HCC into MC and IM, we compared pairs of multiple HCC samples from 12 patients who were clinically diagnosed MC, 6 patients with IM with regard to molecular aberrations, and 2 patients with pairs of primary HCC and extrahepatic metastasis such as adrenal grand and lung metastasis. Exome sequence showed that 125 mutations (90.6%) per patient with IM were common, which were consistent with the result of a pair of primary HCC and extrahepatic metastasis. On the other hand, 138 somatic mutations per tumor, and no common somatic mutations were identified in the 7 patients with MC. Especially only a few passenger mutations were different between IM specimens suggesting that IM pairs developed from the common ancestor. In contrast, 5 patients who clinically diagnosed MC had common mutations (41.7%), which indicates that pairs of HCC of these patients were genetically IM. Notably, mutation signatures obtained from exome sequence data of 2 nodules from the same patients with IM were similar, while those from patients with MC were different by profiling analysis. On the other hand, methylation profile shows that 2 HCC samples from the same patients with MC as well as IM were classified into the same cluster; the methylation status from the different ancestor is similar, and not altered through carcinogenesis. Taken together, comparison of mutations in a pair of HCCs makes it possible to classify multiple HCCs into MC and IM, which is difficult to be correctly determined in clinical practice, and is available to make treatment plans for multiple HCC patients. Furthermore, methylation status of 2 nodules of MC from same patient were similar, while those of mutation signatures were different, which suggests that the epigenomic changes are the earlier event prior to the genomic alterations.

Citation Format: Yutaka Midorikawa, Shogo Yamamoto, Kenji Tatsuno, Hiroki Ueda, Shingo Tsuji, Genta Nagae, Tadatoshi Takayama, Hiroyuki Aburatani. Genetic difference between multicentric carcinogenesis and intrahepatic metastasis of hepatocellular 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 163.
Abstract 1635: The histone demethylase KDM3A in Ewing Sarcoma metastasis
Authors: Sechler Marybeth , Parrish Janet , Jedlicka Paul
Published: July 2016

This study aims to explore the roles and mechanisms of new candidates for therapeutic targeting in Ewing Sarcoma. Ewing Sarcoma is an aggressive pediatric malignancy of the bone and soft tissue, driven by EWS/Ets oncogenic fusions and characterized by high propensity for metastasis and poor clinical outcomes. We have identified the histone demethylase KDM3A as a novel tumor and metastasis promoter downstream of EWS/Fli1, the most common driver in this disease. Moreover, we have identified the cell surface protein, Melanoma Cell Adhesion Molecule (MCAM), as a potential important mediator of KDM3A action. Our data show that both KDM3A and MCAM depletion can inhibit growth and metastatic phenotypes. Additionally we explore the regulation of MCAM, showing that KDM3A may upregulate MCAM expression both through a direct as well as an indirect mechanism via the Ets1 transcription factor. Since both KDM3A and MCAM are pharmacologically targetable, understanding of this pathway could provide at least two new therapeutic targets in Ewing Sarcoma, and, importantly alternatives to drugging of EWS/Ets fusions, which to date has proven very difficult.

Citation Format: Marybeth Sechler, Janet Parrish, Paul Jedlicka. The histone demethylase KDM3A in Ewing Sarcoma metastasis. [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 1635.
Abstract 1645: Tumor-associated macrophages enhance DNA damage repair and improve survival of murine breast cancers after irradiation
Authors: Soto Luis A. , Rafat Marjan , Vilalta Colomer Marta , Giaccia Amato , Graves Edward
Published: July 2016

Tumor-associated macrophages (TAMs) are known to promote physiological processes that drive tumor progression and survival, including angiogenesis, immunosuppression, and invasion. While most studies on TAMs have focused on the molecular mechanisms by which TAMs drive these processes, less focus has been given to direct effects that TAMs may exert on tumor cells. It is known that monocytes are recruited to tumor sites after radiotherapy and support tumor regrowth. In this study we sought to determine if TAMs are able to exert a direct, protective effect on tumor cells against radiotherapy in vitro. We used murine 4T1 cells, a well-characterized animal model for breast cancer, and murine RAW264.7 macrophages to carry out co-culture experiments. Co-culture of 4T1 cells with RAW264.7 macrophages resulted in increased 4T1 cell survival rates post-irradiation compared to 4T1 cells cultured alone. We further determined that this effect is not contact-dependent but mediated through macrophage secreted factors. Next, we tested whether co-culturing RAW264.7 macrophages with 4T1 cells induced macrophage polarization to an M2/TAM phenotype. Using antibodies against M2 markers and FACS, we found that co-culture of RAW macrophages with 4T1 cells polarizes these macrophages toward an M2 phenotype. We hypothesized that the increased 4T1 cell survival rates were mediated by an enhanced DNA damage repair mechanism induced by TAMs. We tested this via a combination of immunofluorescence against phosphorylated histone H2AX and comet assays. We show that co-culture of 4T1 cells with TAMs after irradiation results in lower 4T1 cell levels of DNA damage and a faster decrease in DNA damage over time compared to 4T1 cells alone. Future work will focus on characterizing the molecular mechanism through which TAMs induce this enhanced DNA damage repair response in 4T1 cells after irradiation and we will test whether such response occurs in vivo after radiotherapy. In conclusion, we show that TAMs confer resistance against ionizing radiation to tumor cells in vitro and that this resistance is driven by an enhanced DNA damage repair mechanism in tumor cells induced by TAM secreted factors.

Citation Format: Luis A. Soto, Marjan Rafat, Marta Vilalta Colomer, Amato Giaccia, Edward Graves. Tumor-associated macrophages enhance DNA damage repair and improve survival of murine breast cancers after irradiation. [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 1645.
Abstract 165: Single-cell transcriptome analysis guides tailored combinatorial therapeutics in refractory kidney cancer
Authors: Kim Kyu-Tae , Lee Hye Won , Lee Hae-Ock , Song Hye Jin , Jeong Da Eun , Shin Sang , Kim Hyunho , Shin Yoojin , Nam Do-Hyun , Jeong Byong Chang , Kirsch David G. , Joo Kyeung Min , Park Woong-Yang
Published: July 2016

Metastatic renal cell carcinoma (mRCC) evolves from primary RCC (pRCC) and harbors multiple subpopulations with distinct molecular and phenotypic features. Such underlying intratumoral heterogeneity (ITH) imposes difficulties in designing marker-based clinical trials because targeted mono-therapy eliminates a specific subpopulation of tumor cells while leaving others unharmed. Accordingly, a rational combination strategy that minimizes the survival of the drug-resistant subpopulation in a given heterogeneous tumor is essential for long-term therapeutic efficacy. Here, we examined the ITH of a paired mRCC and pRCC using single-cell mRNA sequencing (scRNA-seq) to identify specific tumor cell populations with drug target pathway activation. From the single cell expression profiles, we found the highly activated status of the EGFR and Src signaling pathways in the mRCC compared to the pRCC, with supporting in vitro high-throughput drug screening results. Distinct features of intratumoral expression variability across mRCC single cells that were masked in the bulk measurement prompted us to test the co-targeting strategy for the EGFR and Src pathways with increased likelihood for complete response. This combinatorial strategy showed significantly better treatment effects on mRCC-derived xenograft platforms in vitro and in vivo than monotherapies. Taken together, our findings show clinical implications of scRNA-seq in designing effective treatment regimens for overcoming treatment failure to conventional monotherapies, and also provide novel insights to the unmet clinical needs in effective personalized treatments.

Citation Format: Kyu-Tae Kim, Hye Won Lee, Hae-Ock Lee, Hye Jin Song, Da Eun Jeong, Sang Shin, Hyunho Kim, Yoojin Shin, Do-Hyun Nam, Byong Chang Jeong, David G. Kirsch, Kyeung Min Joo, Woong-Yang Park. Single-cell transcriptome analysis guides tailored combinatorial therapeutics in refractory kidney 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 165.
Abstract 1652: P16INK4a over-expression sensitizes HPV(-) HNSCC to radiation through down-regulation of USP7
Authors: Molkentine David , Wang Li , Bridges Kathleen , Mason Kathy , Meyn Raymond , Skinner Heath D.
Published: July 2016

Background: Head and neck squamous cell carcinoma (HNSCC) tumorigenesis induced by human papillomavirus (HPV) shows a greater clinical response to radiation therapy when compared to HPV negative patients. The underlying mechanism for this more favorable outcome is unknown, but the presumption is that DNA repair response must play a large role. P16INK4a is a known surrogate marker for HPV positivity; however the relationship between radiation sensitivity and P16INK4a has not been completely elucidated in this context. Ubiquitin specific protease 7 (USP7), also known as HAUSP, is a deubiquitylating enzyme that cleaves ubiquitin from substrates to stabilize target proteins. USP7 overexpression is a predictor of poor prognosis in lung carcinomas and correlates with disease progression and lower survival in gliomas. More recent studies have shown USP7 has a role in the stability of DNA damage response proteins such as RNF168, BRCA1 and Chk1; and USP7 knockdown has been shown to radio-sensitize breast cancer cells to radiation. We have investigated the possible role of USP7 in the radio-response of HNSCC cells in the context of HPV status.

Methods: Four HPV(+) and four HPV(-) HNSCC were chosen for this study. The radio-response was determined by clonogenic survival assay. P16INK4a overexpression was generated using lentivirus. Western blot analysis was used for visualizing protein expression. The Cancer Genome Atlas was interrogated to examine the relationship between USP7 and clinical outcome.

Results: Our data suggest P16INK4a negatively regulates USP7 and leads to the more radiosensitive phenotype associated with HPV(+)HNSCC. Radio-resistant HPV(-) HNSCC cell lines show higher levels of USP7 than HPV(+) lines and over-expression of P16INK4a in HPV(-) HN5 cells leads to down-regulation of USP7 and radio-sensitization. Conversely, P16INK4a siRNA knockdown in HPV(+) UMSCC-47 shows an increase of USP7 protein expression and radio-resistance. P22077, an inhibitor of USP7 activity, was also shown to radiosensitize a HPV(-) cell line. This shows proof of principle that inhibiting USP7 can be a viable approach to sensitizing HPV(-) HNSCC to radiation. USP7 overexpression was also associated with poorer overall survival in HNSCC.

Conclusion: These results suggest that USP7 may be a marker of clinical radioresistance in HNSCC and that its inhibition has the potential to improve the treatment outcome of patients with HPV(-) HNSCC when combined with radiotherapy.

Citation Format: David Molkentine, Li Wang, Kathleen Bridges, Kathy Mason, Raymond Meyn, Heath D. Skinner. P16INK4a over-expression sensitizes HPV(-) HNSCC to radiation through down-regulation of USP7. [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 1652.
Abstract 1656: Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation
Authors: Lee Chen-Ting , Zhou Yingchun , Choudhury Kingshuk R. , Reihani Sharareh Siamakpour , Dewhirst Mark W. , Horton Janet K.
Published: July 2016

Breast cancer is the most common malignancy diagnosed among women worldwide and represents a heterogeneous group of subtypes. Radiation therapy is an important component of multimodal treatment for women with breast cancer. However, little is known about radiation response among these subtypes. Recent clinical data suggests that distinct patterns of response to systemic therapy may exist in association with each phenotype. Therefore, understanding the intrinsic variation of radiation response in breast cancer subtypes is important in prescribing individualized radiotherapy that maximizes the therapeutic ratio.

Our previous studies have identified marked changes in gene expression in breast patient tumors and breast tumor cell lines in response to radiation. Among candidate genes, FAS was consistently and significantly induced after radiation in luminal breast tumors and cell lines. Modulation of FAS in basal breast cancer cell lines favorably impacted radiation response. In this study, we further investigated the role of FAS modulation and radiation response in a mouse model of breast cancer. MCF7 (luminal), HCC1954 (HER2+) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of nude mice. These mice were then treated with different doses of radiation. Our results showed that radiation treatment inhibited MCF7, and to a less extent HCC1954 tumor growth in a dose-dependent manner, while SUM159 tumors were resistant to radiation. The estimated TCD50 value was 19.30 Gy for MCF7 and 44.88 Gy for SUM159. FAS was induced in MCF7 tumors after radiation but showed no change in SUM159 tumors. To investigate whether FAS modulation can affect radiation response, we silenced FAS in MCF7 tumors and activated FAS in SUM159 tumors. We found that silencing of FAS did not negatively impact radiation response in MCF7 tumors, possibly due to compensation by other apoptotic pathways. On the other hand, FAS activating antibody in combination with radiation delayed SUM159 and HCC1954 tumor growth (p = NS).

In conclusion, these results suggest that there is intrinsic variation in radiation response among breast cancer subtypes. FAS concurrent with radiation slows tumor growth in the radiation resistant subtypes, but the effect was not significant. Alternative subtype-specific modulators of radiation response are under investigation.

Citation Format: Chen-Ting Lee, Yingchun Zhou, Kingshuk R. Choudhury, Sharareh Siamakpour Reihani, Mark W. Dewhirst, Janet K. Horton. Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation. [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 1656.
Abstract 1667: A novel method to identify radiosensitizers targeting hypoxic cancer cells by high-throughput screening
Authors: Tamari Keisuke , Seo Yuji , Takahashi Yutaka , Suzuki Osamu , Isohashi Fumiaki , Yoshioka Yasuo , Ogawa Kazuhiko
Published: July 2016

Tumor hypoxia correlates radioresistance and poor clinical outcomes after radiotherapy. Radiosensitizers have been studied for a long time especially to overcome the problem of hypoxic tumor cells. Until now, however, radiosensitizers for hypoxic tumors have had only limited success in clinic. High throughput screening is popular method to identify candidate compounds from a large number of compounds for drug discovery, but have not been established method to study new radiosensitizers. The aim of this study was to identify radiosensitizers targeting hypoxic cancer cells by high throughput screening and validate the efficacy of this method. We performed cell-based high throughput screening by using 384 well plates and 1280 compounds. After exposing compounds to HeLa cells, gamma-irradiation was delivered under hypoxic condition and incubated for 4 days. To identify candidate compounds, cell counting was performed by MTS assay. Then, radiosensitizing effect of these compounds was validated by clonogenic survival assay. We performed high throughput screening and identified 22 compounds as candidates. We selected Ro90-7501 and dicyclomine hydrochloride which were not reported as radiosensitizers. Clonogenic survival assay showed their significant radiosensitizing effect in hypoxic condition. In conclusion, we identified novel radiosensitizers by high throughput screening. This method may be useful for identifying radiosensitizers targeting hypoxic cancer cells.

Citation Format: Keisuke Tamari, Yuji Seo, Yutaka Takahashi, Osamu Suzuki, Fumiaki Isohashi, Yasuo Yoshioka, Kazuhiko Ogawa. A novel method to identify radiosensitizers targeting hypoxic cancer cells by high-throughput screening. [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 1667.
Abstract 1671: Local release of combretastatin A-4 from NIR-light activatable prodrugs overcomes areal and temporal limitations of photodynamic therapy
Authors: Rajaputra Pallavi , Bio Moses , Nkepang Gregory , Thapa Pritam , Woo Sukyung , You Youngjae
Published: July 2016

A unique prodrug strategy for treating localized cancers, in which NIR light-illuminated prodrug effectively ablates tumors through the combined effects of photodynamic therapy (i.e., singlet oxygen [SO]) and locally released anticancer drugs has been proposed. Due to short distance of action (< 0.04 μs) and short lifetime (< 0.02 μm) of SO, direct damage of PDT is both areally and temporally limited. We hypothesized that the locally released anticancer drugs would overcome the areal and temporal limits of SO. Near IR-activatable prodrug of combretastatin A-4 (CA4), Pc-(L-CA4)2, and its pseudo-prodrug, Pc-(NCL-CA4)2, were evaluated in vitro and in vivo. After partial illumination of a 24 well, all the cells in the prodrug-treated well were killed by the released CA4. Limited areal damage was observed in the pseudo-prodrug-treated wells. A time-dependent cell survival study revealed more extensive cell death in the prodrug-treated cells, due to the sustained damage from the released CA4. Cell cycle analysis and microscopic imaging data demonstrated the typical damage patterns of CA4 in the prodrug-treated cells. A time-dependent histological study showed that prodrug-treated tumors lacked mitotic bodies. The prodrug caused broader and more long-lasting tumor size reduction than did the pseudo-prodrug. These data consistently support that the released CA4 overcomes the areal and temporal limits of SO, providing far superior antitumor effects.

Citation Format: Pallavi Rajaputra, Moses Bio, Gregory Nkepang, Pritam Thapa, Sukyung Woo, Youngjae You. Local release of combretastatin A-4 from NIR-light activatable prodrugs overcomes areal and temporal limitations of photodynamic therapy. [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 1671.
Abstract 1672: Photodynamic therapy using photosensitizer and light emitting diodes (LEDs) demonstrates oncolytic activity against prostate cancer
Authors: Gheewala Taher , Skwor Troy , Munirathinam Gnanasekar
Published: July 2016

Prostate cancer (PCa) affects over 2 million Americans and its treatment options include surgery, anti-hormonal drugs for androgen sensitive tumors, and radiotherapy. However, patients undergoing androgen deprivation therapy develop resistance leading to development of castration resistant or androgen independent PCa. It is clear there is an urgent need to find an effective treatment strategy to manage aggressive PCa. An alternative treatment is the use of photodynamic therapy (PDT), which involves the activation of a photosensitizer by a defined wavelength of light in the presence of oxygen, generating transient concentrations of reactive oxygen species. We hypothesize that PDT via a photosensitizer (pheophorbide) in combination with light emitting diodes (LEDs) [670 nm] will demonstrate oncolytic activity against PCa cells, thus suggesting an alternative, less toxic cancer treatment. To test our hypothesis, we used C4-2 and DU-145 PCa cell lines as an in vitro model in this study. To explore the anti-cancer effects of PDT on PCa, cell viability assay and wound healing assay were performed. Western blotting was employed to identify the signaling molecules modulated by PDT. Our cell viability assay results demonstrated significant oncolytic activity against PCa cell lines using PDT with pheophorbide as a photosensitizer combined with 670 nm LEDs in as little as an 88 second pulse with increased lytic activity correlating with elevated energy intensities. We also observed variability in cell line PDT susceptibility; wherein, C4-2 cells were more susceptible than DU-145 PCa cells. The wound healing assay results with pheophorbide and 670 nm LEDs demonstrated significant inhibition of migration abilities of both C4-2 and DU-145 PCa cells. These findings implicate the anti-metastatic activity of PDT on PCa. Further, our mechanistic analysis showed activation of BiP/GRP78, an endoplasmic reticulum (ER) chaperone, in C4-2 as well as DU-145 PCa cells. This suggests that the anti-cancer activity of PDT with pheophorbide and 670 nm LEDs is via ER stress. In conclusion, our results show PDT using pheophorbide and 670 nm LEDs as a promising therapeutic strategy for PCa.

Citation Format: Taher Gheewala, Troy Skwor, Gnanasekar Munirathinam. Photodynamic therapy using photosensitizer and light emitting diodes (LEDs) demonstrates oncolytic activity against prostate 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 1672.
Abstract 1675: Activation of phosphatase toward the retinoblastoma protein inhibits invasion in MDA-MB-231 breast cancer cells
Authors: Egger Jacklynn , Lane Maria , Dedi Brixhilda , Krucher Nancy A.
Published: July 2016

Excessive phosphorylation of the Retinoblastoma protein (Rb) is found in most cancer tumor types. Several cyclin dependent kinase (cdk) inhibitors are in development or in clinical trials at the present time. Our previous studies have focused on the regulation of Rb phosphorylation by the phosphatase, PP1. In proliferating cells, PP1 is regulated by a protein called PNUTS (Phosphatase Nuclear Targeting Subunit). PNUTS is a competitive inhibitor of Rb binding to PP1 and siRNA mediated knockdown of PNUTS in several cancer cell types (breast, colon, ovarian) leads to an increase in Rb directed PP1 activity, Rb dephosphorylation on several sites, and a dramatic induction of apoptosis. In this study we have used MDA-MB-231 breast cancer cells grown in Matrigel. By lentiviral-mediated doxycycline inducible expression of PNUTS siRNA, expression of PNUTS protein is blocked and phosphorylation of Rb cells is reduced. Cells become less invasive and expression of mesenchymal markers N-cadherin, Zeb and Slug is lost. A reversion to the epithelial phenotype is suggested by an increase in E-cadherin expression. PNUTS knockdown has no effect on cells that do not contain highly phosphorylated Rb such as non-transformed MCF-10A cells or Rb-negative MDA-MB-468 cells. Further experiments using highly invasive HT-1080 cells show that reduction of Rb phosphorylation appears to reduce invasiveness in this cell model, supporting the idea that Rb phosphorylation plays a critical role in the regulation of invasion.

Citation Format: Jacklynn Egger, Maria Lane, Brixhilda Dedi, Nancy A. Krucher. Activation of phosphatase toward the retinoblastoma protein inhibits invasion in MDA-MB-231 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 1675.