bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1909: NR4A2 role in head and neck cancer: Mechanistic and functional analysis
Authors: Chaudhary Sanjib , Pothuraju Ramesh , Atri Pranita , Rachagani Satyanarayana , Batra Surinder K. , Macha Muzafar A.
Published: August 2018

Background: Nuclear receptor related 1 protein (NURR1/NR4A2) is a transcription factor that mediates numerous functions in differentiation/development, maintenance and metabolism. In cancer cells, NR4A2 promotes proliferation, migration, transformation and chemo-resistance in many cancers. Here, we explore the role of NR4A2 in head and neck squamous cell cancers (HNC) tumorigenesis and define its target genes. Methods: NR4A2 expression was determined by immunohistochemistry (IHC) and in publically available databases (Oncomine, TCGA). NR4A2 was knockedout/knockdown by CRISPR or shRNA. Coimmunoprepitation and reverse chromatin immunoprecipitation were done to study the interaction between EGFR/STAT3. ChIPSeq analysis was done by using Illumina NextSeq 500 Genome Analyzer and integrated with microarray (HPV+/- patients) to identify the true target genes of NR4A2 using BETA Software and validated by PCR/qPCR. Results: IHC and database analysis demonstrated overexpression of NR4A2 in HNC patients. NR4A2 was significantly overexpressed in HPV+ HNC patients, SCC47 and SCC104 cell lines. Functionally, CRISPR/shRNA knockout/knockdown of NR4A2 in cell lines significantly decreased colonogenicity, proliferation and migration. Expression of NR4A2 was positively correlated with EGFR expression (R2=0.92), and EGF treatment (100ng/ml; 24-48h) induced NR4A2 (~2 fold) expression coupled with epithelial to mesenchymal transition in SCC1 and SCC10B cells (HPV-). We also observed EGF mediated NR4A2 induction was abrogated by panEGFR inhibitor (afatinib) with concomitant decrease in STAT3 expression. Physical interaction between EGFR and STAT3 with EGF treatment regulated NR4A2 expression. Whole genome integration of ChIPseq and microarray demonstrates 9% (2015) and 23% (3002) of the enriched up and down-regulated target genes, respectively were canonically regulated in HPV+ HNC. ChIP PCR validation indicates enrichment of NR4A2 in some of the target genes, wnt10, p53, cxcl13, cln10 and hoxb8 in HPV+ cells. Conclusions: Our data indicate overexpression of NR4A2 in HNC, and its positive correlation with EGFR expression. We also noted the physical interaction between EGFR and STAT3 which functionally attribute the NR4A2 expression. Integration and validation data showed the differential enrichment of NR4A2 target genes in HPV+/- cell lines which suggest its possible discriminatory role in HNC pathogenesis which needs to be confirmed.

Citation Format: Sanjib Chaudhary, Ramesh Pothuraju, Pranita Atri, Satyanarayana Rachagani, Surinder K. Batra, Muzafar A. Macha. NR4A2 role in head and neck cancer: Mechanistic and functional analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1909.
Abstract 1914: Elongation factor 2: A novel target for cancer progression
Authors: Malacrida Beatrice , Dowling Catríona M. , Flygaard Rasmus K. , Kiely Maeve , Coffey John C. , Jenner Lasse B. , Kiely Patrick A.
Published: August 2018

Protein translation is one the most energy demanding and highly regulated processes in cells. For this reason, dysregulation of protein synthesis is linked with different types of disease, including cancer. The link between protein synthesis and cancer progression is becoming a central theme among cancer research. Although numerous studies have identified protein synthesis as a process central to cancer progression, further research is required to identify potential novel targets and/or biomarkers.

Recently, RACK1 (Receptor for Activated C Kinase 1), a scaffold protein involved in many cell regulatory pathways including cell proliferation and migration, has been found to be part of the ribosomal machinery. Here, RACK1 is able to regulate the synthesis of particular sets of proteins and the translation itself. In fact, RACK1 is located close the mRNA exit channel where is able to recruit kinases, phosphatases and specific mRNA binding proteins.

We have identified elongation factor 2 (eEF2) as novel RACK1 interacting protein in our two and three dimensional colon cancer models. Elongation factor 2 is one of the main proteins regulating the elongation phase of protein synthesis, responsible for the translocation of the mRNA along the ribosome. We have confirmed the binding between eEF2 and RACK1 by isolating and purifying a native human form of eEF2 and combining it with a purified human version of RACK1.

Interestingly, we have demonstrated that stress conditions and IGF-1 stimulation modulate eEF2 activity in colon cancer cells. Moreover, we have examined the expression of EEF2 and have found that EEF2 gene is dysregulated in our cohort of matched normal and cancer colonic patient tissues. Interestingly, EEF2 expression is upregulated in mucinous cancers (a rare and aggressive type of colorectal cancer) compared to non-mucinous cancers and the expression of EEF2 is as well upregulated in cancers that have recurred within 2 to 3 years after the first surgery.

We believe that eEF2 is playing an important role in cancer progression and migration especially by regulating and modulating protein synthesis in certain conditions. A better understanding of the role of this protein will help in the identification of novel targets that can be then translated into therapeutic strategies against colorectal cancer.

Citation Format: Beatrice Malacrida, Catríona M. Dowling, Rasmus K. Flygaard, Maeve Kiely, John C. Coffey, Lasse B. Jenner, Patrick A. Kiely. Elongation factor 2: A novel target for cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1914.
Abstract 1919: Characterization of URST1 as a prognostic biomarker and therapeutic target for oral cancer
Authors: Takano Atsushii , Miyagi Yohei , Daigo Yataro
Published: August 2018

Oral cancer shows a high mortality rate, and its incidence is increasing gradually worldwide. Current therapies for oral cancer include surgery, radiotherapy, and chemotherapy, however, local and regional recurrences account for up to 90% of treatment failures after surgery and radiotherapy. Therefore, next generation biomarkers and therapeutic strategies for oral cancer are eagerly awaited. To identify biomarkers and therapeutic targets for oral cancer, we screened genes that overexpressed in the majority of oral cancers by using our original gene expression profile database and identified up-regulated in solid tumor 1 (URST1) as a candidate. Immunohistochemical staining showed that URST1 expression was observed in the majority of oral cancer in 64 of 96 (67%) that had received curative surgery. URST1 expression was associated with poor prognosis for oral cancer patients (P = 0.032 by log-rank test). Reduction of URST1 expression by siRNAs against URST1 (si-URST1) significantly suppressed growth of oral cancer cells. Flow cytometric analysis revealed that si-URST1 increased the population of cancer cells at G2/M. In addition, exposure of cancer cells to selective URST1 inhibitor suppressed the growth of oral cancer cells. These data indicate that URST1 is a possible prognostic biomarker and therapeutic target for oral cancer.

Citation Format: Atsushii Takano, Yohei Miyagi, Yataro Daigo. Characterization of URST1 as a prognostic biomarker and therapeutic target for oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1919.
Abstract 1920: Targeting structural RET and MET kinase alterations in lung adenocarcinoma patients
Authors: Plenker Dennis , Lorenz Carina , Bertrand Miriam , Riedel Richard , Langen Joop de , Brägelmann Johannes , Büttner Reinhard , Wolf Jürgen , Thomas Roman K. , Heuckmann Johannes , Sos Martin L.
Published: August 2018

Structural rearrangements that activate receptor kinases account for an ever-increasing pool of druggable targets in lung adenocarcinoma patients. Among these tumors RET kinase fusions and MET alterations represent two genetically distinct groups that share a common lack of clinically effective strategies. While RET rearranged tumors show a limited susceptibility to currently available RET inhibitors, MET rearranged tumors have not been yet fully appreciated as a relevant group that may benefit from treatment with MET targeted drugs. Using systematic molecular profiling of genetically engineered RET rearranged in vitro and models and patient-derived in vivo models we identified the type II kinase inhibitors ponatinib and AD80 as the most potent drugs. While both inhibitors are effective against gatekeeper mutant RET we identified a novel resistance mutation RETI788N that triggers a selective resistance against AD80 and other RET inhibitors but retains susceptibility to ponatinib. Furthermore, we studied the clinical and preclinical activity of MET targeted drugs. We specifically characterized genomic rearrangements of KIF5B-MET and STARD3NL-MET in cellular models that were found in two distinct LADC. In parallel we identified and characterized a MET kinase domain duplication that developed in an EML4-ALK rearranged positive tumor as a resistance mechanism to ceritinib. All three patients showed a partial response to crizotinib that effectively inhibits MET and ALK in these tumors. Thus, our molecular characterization of drug-target engagement in genetically defined models may further enhance the clinical efficacy of kinase inhibitors in lung tumors driven by rare oncogenic kinase alterations.

Citation Format: Dennis Plenker, Carina Lorenz, Miriam Bertrand, Richard Riedel, Joop de Langen, Johannes Brägelmann, Reinhard Büttner, Jürgen Wolf, Roman K. Thomas, Johannes Heuckmann, Martin L. Sos. Targeting structural RET and MET kinase alterations in lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1920.
Abstract 1923: Identification and validation of Integrin-Linked Kinase (ILK) signaling as a therapeutic target for ovarian cancer
Authors: Ulm Michael A. , Ponnusamy Suriyan , ElNaggar Adam C. , Narayanan Ramesh
Published: August 2018

Ovarian cancer affects approximately 25,000 women and contributes to around 14,000 deaths in the United States each year. Although significant advances have been made in the discovery of molecular diagnostics and therapeutics for other cancers, ovarian cancer still lacks reliable diagnostic and prognostic markers as well as appropriate targeted therapeutics. A major reason for the lagging development of targeted therapeutics is the molecular heterogeneity of high grade serous ovarian cancer and the lack of better understanding of driver mutations and pathogenesis. Although gene expression data sets are available, most of them lack appropriate pair-matched controls to define the alterations that result in the transformation of normal ovarian cells to highly proliferative cancerous cells. We compared differential gene expression of high-grade treatment-naïve ovarian cancer tissue samples to matched-pair adjacent benign ovarian tissue specimens from 24 patients. A significant upregulation of the integrin-linked kinase (ILK) pathway was identified in 22 of the 24 cancer specimens, identifying a potential player that could contribute to the transformation of normal ovarian cells to cancerous cells. To determine the therapeutic potential of ILK inhibition, ILK expression was reduced using siRNA in SKOV3 cells, which resulted in decreased cell proliferation. This is consistent with prior studies demonstrating reduced adhesion to and invasion of collagen gels and organotypic meso-mimetic ovarian cancer cultures with down-regulating ILK using siRNA resulted in. We then assessed the ability of a targeted agent, compound 22, a small molecule ILK-selective inhibitor, to inhibit proliferation in various ovarian cancer cell lines with different phenotypic characteristics (SKOV3, OVCAR3, OVCAR8, OV90). Compound 22 reduced the phosphorylation of Akt, a downstream target of ILK, and also inhibited cellular proliferation. Our initial findings validate ILK as a potential therapeutic target for molecular inhibition in ovarian cancer and warrants further investigation.

Citation Format: Michael A. Ulm, Suriyan Ponnusamy, Adam C. ElNaggar, Ramesh Narayanan. Identification and validation of Integrin-Linked Kinase (ILK) signaling as a therapeutic target for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1923.
Abstract 1931: Targeting master neuronal transcription factor BRN2 in neuroendocrine prostate cancer
Authors: Thaper Daksh , Munuganti Ravi , Norouzi Shaghayegh , Kumar Sahil , Kim Soojin , Singh Kriti , Vahid Sepideh , Bishop Jennifer , Zoubeidi Amina
Published: August 2018

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges and patients generally die within two years. In particular, a subset of patients who relapse following ARPI therapy exhibit lineage switching whereby tumours shed their dependence on AR signaling and emerge with neuroendocrine features. These tumours, termed treatment induced neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapy which carries a short-lived response at the cost of significant toxicity. Therefore, targeted therapies for this deadly disease are desperately needed. Thus, the need to develop targeted treatments for this devastating disease is of paramount importance. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of NEPC and aggressive tumor growth, both in vitro and in vivo, suggesting targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC. Methods: Study the effects of BRN2 inhibition using siRNA and CRISPR K/O models. Results: Inhibition of BRN2 by siRNA and by CRISPR/Cas9 knockout drastically reduced cell proliferation in 42DENZR (NEPC) cell lines. This data was re-capitulated in human NEPC NCI-H660 cells. Loss of BRN2 initiated drastic epigenetic changes in NEPC cell lines as well as in G1 arrest through up-regulation of CDKN1A/1B. This was confirmed using our first in field BRN2 inhibitors. Targeting BRN2 also lead to downregulation several known targets in NEPC like EZH2, AURKA, SOX2 and Peg10. Conclusion: No therapies exist for highly lethal NEPC. Hence, the described work aims to verify BRN2 as a central driver of NEPC, and lay the pre-clinical foundation for the integration of targeted therapies into the treatment landscape to improve survival and quality of life for patients suffering from deadly form of prostate cancer.

Citation Format: Daksh Thaper, Ravi Munuganti, Shaghayegh Norouzi, Sahil Kumar, Soojin Kim, Kriti Singh, Sepideh Vahid, Jennifer Bishop, Amina Zoubeidi. Targeting master neuronal transcription factor BRN2 in neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1931.
Abstract 1945: Degradation of ERG transcription factor as a targeted therapy in B-cell and T-cell acute lymphoblastic leukemia
Authors: Wang Xiaoju , Cheng Caleb , Qiao Yuanyuan , Xiao Lanbo , Wang Cynthia , Parolia Abhijit , Chinnaiyan Arul
Published: August 2018

Acute lymphoblastic leukemia (ALL) occurs in children and adults and is one of the most common forms of pediatric cancer. The ideal individualized therapy for B- and T-cell ALL heavily depends on the present molecular drivers. However, even with the success of ALL therapy, only a few strategies target disease-specific molecular lesions. Philadelphia chromosome positive (Ph+) ALL, which harbors the BCR:ABL1 rearrangement, are sensitive to tailored inhibitors. Unfortunately, most ALL cases in both children (97-98%) and adults (70-80%) are Ph-; therefore, agents designed to directly target other molecular lesions are urgently needed. ERG, an ETS-family transcription factor, plays a key role in hematopoietic differentiation, megakaryopoiesis, and megakaryoblastic leukemia associated with Down syndrome. ERG overexpression is predictive of a poor prognosis in acute myeloid leukemia and T-ALL. Transgenic expression of ERG causes T-ALL in mice and knockdown of ERG reduces human T-ALL cell proliferation. We have previously identified a consensus peptide (EIPs) that specifically binds to and leads to the proteosomal degradation of the ERG protein in prostate cancers having the TMPRSS2:ERG gene fusion. Here, we demonstrated that wild type ERG was overexpressed at the mRNA and protein levels in a subset of B- and T-cell ALLs, but not in precursor CD4+, CD8+, or CD19+ cells. Pull-down assay found that streptavidin-linked beads were enriched for ERG proteins. Treatment of EIPs induced proteolytic degradation of ERG protein in Hal-01 cells, and therefore selectively inhibited ERG-driven cell growth and proliferation. More importantly, structure-based optimization created the next generation of ERG degraders (EIPv2). EIPv2 treatment significantly decreased ERG protein in Hal-01 cells starting from 3 hrs and nearly depleted ERG proteins at 48 hrs, resulting in 10-fold more potency than EIPs (IC50=2.3µM vs 25.7µM for EIPv2 and EIPs, respectively). Together, our results validate the development of peptidomimetics for ERG depletion as a targeted therapy for ALL. Unlike small-molecule antagonists, which require continuous dosing that eventually induces drug resistance, most protein degraders have relatively higher and longer pharmacological effects without requiring continuous exposure to high doses. Such an approach can abolish the presence of an oncoprotein, which, in theory, can overcome resistance to target inhibitors.

Citation Format: Xiaoju Wang, Caleb Cheng, Yuanyuan Qiao, Lanbo Xiao, Cynthia Wang, Abhijit Parolia, Arul Chinnaiyan. Degradation of ERG transcription factor as a targeted therapy in B-cell and T-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1945.
Abstract 1951: Role of mitochondrial peptide Humanin in the response of experimental breast cancer to chemotherapy
Authors: Zuccato Camila , Ayala Mariela A. Moreno , Gottardo Maria F. , Pidre Matias Luis , Asad Antonela S. , Candia Alejandro J. Nicola , Romanowski Victor , Seilicovich Adriana , Candolfi Marianela
Published: August 2018

Humanin (HN) is a mitochondrial-derived peptide with potent cytoprotective effect in many cell types. Administration of HN has been proposed as a therapeutic approach for several chronic diseases, such as diabetes, neurodegenerative conditions and cardiovascular diseases. Although HN has been shown to protect normal tissues against the toxic effects of chemotherapy, its role in tumor chemoresistance is poorly understood. Here, we aimed to elucidate the role of HN in the progression and cytotoxic response of experimental triple negative breast cancer (TNBC). We detected HN expression in murine and human TNBC cells, which was upregulated in the presence of chemotherapeutic drugs, i.e. Doxorubicin (DOXO) and Cisplatin, as assessed by flow cytometry. Addition of HN protected TNBC cells from various cytotoxic insults, such as serum deprivation, proapoptotic cytokine TNF-α or chemotherapy with DOXO. We next evaluated the role of endogenous HN in the apoptotic response of TNBC cells, using a plasmid encoding a short hairpin RNA to inhibit HN expression (pUC.shHN). To readily assess transduction efficiency, the plasmid also encodes the red fluorescent protein dTomato as a reporter gene. Transfection of TNBC 4T1 tumor cells with pUC.shHN increased the percentage of apoptotic cells when compared to cells transfected with control plasmid, as assessed by flow cytometry after staining with propidium iodide. When transfected 4T1 cells were incubated with different concentrations of DOXO, pUC.shHN reduced their clonogenic capacity. We also evaluated the effect of HN in the progression of experimental mouse 4T1 TNBC treated with DOXO. HN was readily detected in tumor samples and lung metastases. Systemic administration of HN reduced tumor apoptotic rate, accelerated tumor progression and increased the development of lung metastases. In addition, administration of HN impaired the antitumor and antimetastatic effect of chemotherapy. In summary, our findings suggest that HN inhibits the response of TNBC cells to cytotoxic stimuli, facilitating tumor progression and chemoresistance. Thus, blockade of HN could constitute a therapeutic strategy to improve the efficacy of chemotherapy in breast cancer.

Citation Format: Camila Zuccato, Mariela A. Moreno Ayala, Maria F. Gottardo, Matias Luis Pidre, Antonela S. Asad, Alejandro J. Nicola Candia, Victor Romanowski, Adriana Seilicovich, Marianela Candolfi. Role of mitochondrial peptide Humanin in the response of experimental breast cancer to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1951.
Abstract 1958: Benztropine enhances temozolomide sensitivity by restricting the growth of drug-resistant glioma stem cells
Authors: Bonnin Damian A. Almiron , Lee Myung Chang , Howard Joseph M. , Young Alison L. , Havrda Matthew C.
Published: August 2018

Glioblastomas (GBMs) are the most aggressive type of primary brain tumor in adults, the second leading cause of cancer-related death in children, and are incurable using currently available treatment modalities. GBMs are characterized by the presence of primitive glioma stem-like cell subpopulations that resist chemotherapy and sustain disease progression. In this study, we found that Benztropine, a muscarinic acetylcholine receptor (mAChR) inhibitor, suppresses glioma cell proliferation and sensitizes these cells to treatment with Temozolomide. Modeling the development of Temozolomide resistance in vitro, we identified a subpopulation of glioma cells, defined by the co-expression of CD44, CD15, and PDGFRa that was resistant to Temozolomide. This chemo-resistant subpopulation was phenotypically similar to oligodendroglial progenitor cells (OPC-like); a stem-like cell population important in the initiation and progression of glioblastoma known to be modulated by mAChR signaling. The addition of the mAChR inhibitor, benztropine, dramatically reduced the emergence of this Temozolomide-resistant sub-population. To begin understanding the mechanism of action of benztropine on GBM cells, we analyzed the effect of this drug on a panel of mitotic drivers that have been implicated in glioma initiation and progression. Sub-lethal doses of Benztropine alone or in combination with temozolomide repressed the PDGF-signaling axis as characterized by a significant reduction of PDGFA secretion and PDGFRA activation. We also developed an in vivo model of Temozolomide resistance to determine whether Benztropine could improve the survival of animals harboring glioma xenografts that had recurred following treatment with Temozolomide. These in vivo studies demonstrated that Benztropine alone improves animal survival as

compared with vehicle controls and that Benztropine combined with Temozolomide reduces glioma growth following initial Temozolomide treatment and improves survival in mice xenografted with human GBM cells. Taken together, our results suggest that Benztropine might be targeting glioma stem cells by altering the glioma stem cell microenvironment. These findings support a potential role for the use of adjuvant Benztropine for the clinical management of GBM.

Citation Format: Damian A. Almiron Bonnin, Myung Chang Lee, Joseph M. Howard, Alison L. Young, Matthew C. Havrda. Benztropine enhances temozolomide sensitivity by restricting the growth of drug-resistant glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1958.
Abstract 1986: IRAK1 augments cancer stemness and drug resistance via the AP-1/AKR1B10 signaling cascade in hepatocellular carcinoma
Authors: Cheng Bowie Yik Ling , Leung Doris Hoi Wing , Lau Eunice Yuen Ting , Ng Irene Oi Lin , Lee Kin Wah
Published: August 2018

Frequent relapse and drug resistance can be attributed to the existence of tumor-initiating cells (T-ICs) within the tumor bulk. From transcriptome sequencing of 16 pairs of clinical HCC samples, we found that Interleukin-1 receptor-associated kinase 1 (IRAK1) in the TLR/IRAK pathway was significantly upregulated in hepatocellular carcinoma (HCC). IRAK1 overexpression in HCC was further confirmed at the mRNA and protein levels and correlated with advanced tumor stages and poor patents survival. Interestingly, IRAK4, an upstream regulator of IRAK1, was also found to be consistently upregulated. We demonstrated that IRAK1 regulates liver T-IC properties, including self-renewal, tumorigenicity and liver T-IC marker expression. IRAK1 inhibition sensitized the HCC cells to doxorubicin and sorafenib treatment in vitro through the suppression of the apoptotic cascade. Pharmacological inhibition of IRAK1 with a specific IRAK1/4 kinase inhibitor consistently suppressed liver T-IC populations. Through RNA sequencing analysis by comparing gene expression profiles between IRAK1-knockdown and control cells, we identified Aldo-Keto Reductase Family 1 Member 10 (AKR1B10) as a novel downstream target of IRAK1. Clinically, AKR1B10 was found to be overexpressed in HCC, which was significantly correlated with IRAK1 expression. Functional analysis demonstrated that knockdown of AKR1B10 negated the IRAK1-induced T-IC functions via modulation of the AP-1 complex. Using an HCC xenograft model, we found that an IRAK1/4 inhibitor in combination with sorafenib synergistically suppressed the tumor growth. In conclusion, targeting the IRAK4/IRAK1/AP-1/AKR1B10 signaling pathway may be a potential therapeutic strategy against HCC.

Citation Format: Bowie Yik Ling Cheng, Doris Hoi Wing Leung, Eunice Yuen Ting Lau, Irene Oi Lin Ng, Kin Wah Lee. IRAK1 augments cancer stemness and drug resistance via the AP-1/AKR1B10 signaling cascade in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1986.
Abstract 2007: A novel CRISPR/Cas9 reporter system to monitor TNBC cancer stem cells in real time
Authors: Truchan Nathan A. , Buschhaus Johanna , Brooks Michael , Halabu Andre , Guo Hongwei , Yuan Hebao , Luker Kathryn , Wicha Max , Sun Duxin , Burnett Joseph P.
Published: August 2018

Increasing evidence suggests that breast cancers arise from so-called cancer stem cells (CSC), which, consistent with their normal adult counterpart, are uniquely capable of long term self-renewal and differentiation into all bulk cancer cell types that exhibit unique epigenetic states within a tumor. While FACS has been instrumental to isolating these cells and quantifying efficacy of CSC targeted drugs in vitro and in vivo, having to remove cells from their native environment is an inherent limitation, thereby only providing a snapshot of information with each experiment. In order to directly visualize CSC dynamics in real time the CRISPR/Cas9 system was used to precisely knockin mCherry directly downstream of the final exon of ALDH1A3 in SUM149 triple-negative breast cancer cells. With regard to CSC properties, ALDH1A3 high cells were shown to be dramatically correlated with the ALDEFLUOR assay and functionally, increased ammosphere and tumor formation in vitro and in vivo. Strikingly, a single ALDH1A3 high cell was capable of reconstituting the entire heterogeneity of the parental cell line. With the ability to directly visualize CSCs we sought to utilize live cell imaging to observe population changes in response to varying therapeutics. As evident by mCherry fluorescence intensity we confirm previous findings that suggest conventional chemotherapeutics such as docetaxel enrich for CSCs while the natural product sulforpahane reduces the population. Taken together, we believe our mCherry reporter cells could be adapted to rapidly screen for novel inhibitors of CSCs in triple-negative breast cancers.

Citation Format: Nathan A. Truchan, Johanna Buschhaus, Michael Brooks, Andre Halabu, Hongwei Guo, Hebao Yuan, Kathryn Luker, Max Wicha, Duxin Sun, Joseph P. Burnett. A novel CRISPR/Cas9 reporter system to monitor TNBC cancer stem cells in real time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2007.
Abstract 2016: Potential role of polo-like kinase 1 in epithelial-mesenchymal transition in melanoma
Authors: Chhabra Gagan , Singh Chandra K. , Ndiaye Mary A. , Ahmad Nihal
Published: August 2018

Mammalian polo-like kinase 1 (Plk1), an important serine/threonine kinase, has been shown to be a critical regulator of mitosis and cytokinesis. A number of studies, including those from our laboratory, have shown that Plk1 is significantly overexpressed in several cancers, including melanoma. Plk1 has also been linked with poor disease prognosis in cancer patients. A limited number of recent studies have suggested that Plk1 may be involved in epithelial-mesenchymal transition (EMT) in different cancers. However, the role of Plk1 in EMT process in melanoma has not been studied. Indeed, EMT plays a driving role in the acquisition of cancer metastasis and an important hallmark of EMT is downregulation of epithelial marker E-cadherin, and upregulation of mesenchymal marker, N-cadherin. In this study, we investigated whether the kinase activity of Plk1 plays a role in EMT process by modulating EMT markers in melanoma cells. To determine this, we performed western blots, RT-qPCR, cell migration and cell invasion assays using A375 melanoma cells stably transfected with plasmids for wild-type (WT) Plk1 overexpression, constitutively active Plk1 (T210D) and kinase-inactive (K82R) Plk1, as well as shRNA-mediated Plk1 knockdown. We observed that overexpression of WT Plk1 upregulated the mesenchymal markers N-cadherin, Vimentin, and Fibronectin, and downregulated the epithelial marker E-cadherin. We also found that the modulation in EMT markers by Plk1 in melanoma cells was associated with the upregulation of transcription factors Snail and Zeb1. Interestingly, downregulation of endogenous Plk1 by specific shRNA resulted in a downregulation of N-cadherin, Vimentin, Fibronectin, Snail and Zeb1. Further, when we assessed the EMT-related molecular changes in A375 melanoma cells containing constitutively active and kinase-inactive Plk1, the cells containing constitutively active Plk1 showed a significant decrease in epithelial marker as well as marked increases in mesenchymal markers. However, the cells containing kinase-inactive Plk1 showed the opposite trend. Moreover, A375 melanoma cells containing constitutively active Plk1 showed higher migration and invasion potential, whereas cells containing kinase-inactive Plk1 showed limited cell migration and invasion. Overall, these results suggest that Plk1 is involved in the EMT process and its kinase activity is important for EMT-related changes in melanoma cells. Further studies are in progress to determine the cause-and-effect molecular mechanisms of Plk1 mediated EMT regulation.

Citation Format: Gagan Chhabra, Chandra K. Singh, Mary A. Ndiaye, Nihal Ahmad. Potential role of polo-like kinase 1 in epithelial-mesenchymal transition in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2016.
Abstract 2027: P62/IMP2 promotes metastasis in hepatocellular carcinoma (HCC) by regulating EMT in different signaling pathway
Authors: Xing Mengtao , Shi Jianxiang , Li Jitian , Chen Beibei , Francia Giulio , Zhang Jianying
Published: August 2018

Insulin- like growth factor 2 mRNA binding proteins (IMPs) Family contains three members: IMP1, IMP2 and IMP3. IMPs are oncofetal proteins, expressed during embryogenesis and lost in most tissues in adults. Overexpressed IMPs have been reported in various types of cancers. They share similar protein structure in mammals and have similar functions: to bind specific mRNAs, stabilize and extend their half-life. IMP2 was first reported as a tumor-associated antigen in HCC. However, only a few research was done on what a role of p62/IMP2 plays on HCC progression.

Previous studies show that IMP2 is overexpressed in HCC tissues and in several human HCC cell lines. Western blotting was performed for different HCC cells has a relatively low expression in SNU449 and higher expression in HepG2 and Hep3b. Stable IMP2 overexpression SNU449 cell line was made by IMP2 cDNA plasmid transfection; stable IMP2 knockout Hep3b and HepG2 cell lines was made by IMP2 Crispr-Cas9 KO plasmid transfection. Wound healing assay has shown that overexpression of p62/IMP2 can significantly enhance cell migration ability in SNU449 cells. On the contrary, the depletion of p62/IMP2 can reduce the migration ability in HepG2 and Hep3b cells (p<0.05). Furthermore, we analyzed HCC gene expression data from TCGA. Median count of IMP2 was used to separate HCC expression data into high and low IMP2 expression group to conduct differential gene expression analysis. Gene Ontology over-representation analysis shows differentially expressed genes (adjusted p<0.05) mainly enriched in membrane part and plasma membrane. The expression of several genes involved in EMT regulation such as CTNNB1, SMAD2, SNAI2, AKT2 shows significant difference (adjust p<0.05). Western blotting assay was performed to show whether IMP2 is involved in EMT regulation. Wnt/beta-catenin pathway and TGF-beta/SMAD pathway were both activated and further upregulated SNAIL, which is an E-cadherin inhibitor. Western blotting results showed that E-cadherin was inhibited in IMP2 overexpressed SNU449 cells, which is supportive to our hypothesis.

Citation Format: Mengtao Xing, Jianxiang Shi, Jitian Li, Beibei Chen, Giulio Francia, Jianying Zhang. P62/IMP2 promotes metastasis in hepatocellular carcinoma (HCC) by regulating EMT in different signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2027.
Abstract 2029: Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells
Authors: Showalter Christian A. , Burdick Monica M.
Published: August 2018

Activation of Notch signaling has been shown to promote cell migration properties and hematogenous metastasis by inducing a dramatic phenotypic change in breast cancer cells via the epithelial-to-mesenchymal transition (EMT). In response to these findings, Notch signaling inhibitors are being explored as therapeutics to prevent EMT in cancers. However, research examining the effects of Notch inhibitors on the activity of sialofucosylated ligands used by circulating tumor cells to adhere to vascular endothelial E-selectin during metastasis is lacking. To advance knowledge in this area, our study investigated the use of an indirect Notch signaling inhibitor (DAPT) on modifying E-selectin ligand activity of breast cancer cell lines by shear flow adhesion assays performed at physiological bone marrow shear stress. DAPT exposure to mesenchymal-like MDA-MB-231 and MDA-MB-468 (induced to the mesenchymal-like state by exogenous epidermal growth factor) breast cancer cells increased E-selectin ligand activity. Furthermore, mesenchymal-like MDA-MB-468 cells after DAPT exposure demonstrated E-selectin ligand activity similar to that of epithelial-like MDA-MB-468 cells. This supports the hypothesis by our group that epithelial-like and mesenchymal-like phenotypes determine activity of functional E-selectin ligands in breast cancer cells, as we have previously shown that epithelial cells display high E-selectin ligand activity while mesenchymal cells display little or no ligand activity. Cell migration ability was assessed using wound closure assays. Mesenchymal-like MDA-MB-468 cells exposed to DAPT exhibited decreased migration compared to mesenchymal-like MDA-MB-468 cells exposed to the DMSO diluent control, whereas mesenchymal-like MDA-MB-231 cells demonstrated no difference in migration compared to DMSO exposed cells. Altogether, our results suggest Notch inhibitors may both increase fluid-shear resistant adhesion and alter migration ability of mesenchymal-like breast cancer cells, and that factors present in the blood might impact the metastatic potential of cancer cells via regulating cellular phenotypes.

Citation Format: Christian A. Showalter, Monica M. Burdick. Notch signaling inhibitor increases E-selectin/ligand interactions and alters cell migration of mesenchymal-like breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2029.
Abstract 2036: Alternative activation of TGFβ signaling pathways in breast cancer tumors with different subtype is related to a differential expression of transcription factors that induce EMT
Authors: Ortega-Hernández Victoria , Fernandez Wanda , Carvallo Pilar
Published: August 2018

Breast cancer is the leading cause of cancer death among women worldwide being distant metastases the main cause of disease. Patients with triple negative breast cancer tumors develop distant metastases earlier compared to luminal tumors. Epithelial-mesenchymal transition (EMT) induced by TGFβ signaling pathways, is the main mechanism to promote metastasis through the expression of the transcription factors TWIST, SNAIL, SLUG and ZEB1. Expression of EMT transcription factors has been studied in cancer cell lines however studies in breast cancer tumors with different subtype have not been reported. In addition, the molecular mechanism that induce EMT program is not known yet. In this study we evaluated the expression of the four transcription factors as well as the state of TGFβ/SMAD, ERK/MAPK and PI3K/AKT pathways in breast cancer cell lines HCC1937 (triple negative) and T47D (luminal) after TGFβ treatment. The same analysis was done by immunohistochemistry in 100 breast cancer tumors with different subtype. After TGFβ treatment T47D showed activation of ERK/MAPK and PI3K/AKT pathways and expression of only two transcription factors: SNAIL and SLUG. HCC1937 cells showed activation of TGFβ/SMAD and ERK/MAPK pathways and expression of TWIST, SLUG and ZEB1 but not SNAIL. Similar results were observed in breast cancer tumors, being TGFβ/SMAD pathway active in the majority of triple negative tumors, whereas an active state of PI3K/AKT was observed in luminal tumors. Active ERK/MAPK pathway was observed in both triple negative and luminal tumors. In relation to expression of transcription factors triple negative tumors with an active state of the TGFβ/SMAD pathway showed expression of transcription factors TWIST, SNAIL and SLUG, whereas those tumors with an active state of ERK/MAPK pathway showed only ZEB1 expression. Luminal tumors with an active PI3K/AKT pathway frequently showed ZEB1 expression. Our results showed that expression of a specific EMT transcription factor induced by TGFβ is related to the tumor subtype and depends on the activated signaling pathway. These results suggest different mechanisms of EMT induction by TGFβ in relation to breast cancer tumor subtype.

Citation Format: Victoria Ortega-Hernández, Wanda Fernandez, Pilar Carvallo. Alternative activation of TGFβ signaling pathways in breast cancer tumors with different subtype is related to a differential expression of transcription factors that induce EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2036.
Abstract 2052: A novel PHD2/VHL- mediated regulation of YAP1 and its role in vascular mimicry and tumor angiogenesis
Authors: Singhal Namrata Bora , Saha Biswarup , Chellappan Srikumar
Published: August 2018

Non-small cell lung cancer (NSCLC) is the leading cause of cancer related death worldwide. In this study, we show that transcriptional co-activator YAP1, the oncogenic component of the Hippo pathway, may contribute to the progression of NSCLC by supporting tumor angiogenesis and vascular mimicry of cancer stem-like cells (CSCs). NSCLC CSCs were found to have higher mRNA expression of VEGF receptor II (KDR) and Angiopoietin-2 (AngPT-2); depletion of YAP1 inhibited the promoter activity as well as the mRNA expression of VEGF, KDR and AngPT-2, with a corresponding reduction in vascular mimicry as well as tumor growth in a mouse xenograft studies. These results suggest a possible mechanism by which YAP1 regulates tumor angiogenesis.

A role for YAP1 in tumor angiogenesis was further supported by the finding that NSCLC cells grown in hypoxic conditions showed higher levels of YAP1. There was little to no changes in the canonical Hippo pathway proteins like LATS1/2, MST1/2, SAV and MOB as well as in the expression of TAZ, an orthologue of YAP1. Elevated YAP1 was found to associate with HIF1α under hypoxic conditions and enhance its transcriptional activity; YAP1 could increase HIF1α-mediated induction of the VEGF promoter confirmed by chromatin immunoprecipitations and transient transfection assays. Elevated levels of YAP1 and HIF1α interaction was detected in lung tumor tissues compared to normal lung tissue, as detected by proximity ligation assay (PLA), suggesting that the higher association of YAP1 with HIF1α and resulting transcriptional activity might have contributed to tumor growth.

An examination of the underlying mechanism by which YAP1 levels are elevated under hypoxic conditions revealed a novel regulation of YAP1 protein by prolyl hydroxylase PHD2 and E3 ubiquitin ligase VHL, which are mainly known to regulate HIF1α under normoxia. PHD2 was found to hydroxylate proline residue(s) of YAP1 between aa 284 to aa 289 as seen by mutational studies. YAP1 was found to directly associate with PHD2 as well as with VHL. Depletion of PHD2 or treatment with DMOG, an inhibitor of prolyl hydroxylases, reduced YAP1 association with VHL ligase. This further elevated YAP1 levels in the nucleus. Interestingly, disruption of the YAP1-PHD2 interaction using a domain specific peptide enhanced the angiogenic tubule formation by endothelial cells. Our data therefore identifies a novel non-canonical pathway of regulation of YAP1 that supports angiogenesis and tumor growth.

Citation Format: Namrata Bora Singhal, Biswarup Saha, Srikumar Chellappan. A novel PHD2/VHL- mediated regulation of YAP1 and its role in vascular mimicry and tumor angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2052.
Abstract 2059: Alcohol enhances estrogen-responsive breast cancer neovascularization
Authors: Cruz Natalie C. , Maniyar Rachana , Suriano Robert
Published: August 2018

The pleiotropic effects of estrogen in breast cancer development have been observed at both the initiation and progression phases. Interestingly, alcohol, as a dietary component, has been observed to possess estrogenic activity, but the synergistic interaction between alcohol and estrogen and its cellular targets remains undefined. Our previous study identified neovascularization as a cellular target of estrogen that was mediated by the mobilization of bone marrow-derived endothelial progenitor cells (BM-EPCs) and their subsequent incorporation within the tumor mass. The present study was undertaken to define the synergistic interaction between estrogen and alcohol using a cell culture model. To examine the effects of both estrogen and alcohol on breast cancer progression, we developed an in vitro model using the murine mammary tumor cell line, TG1-1, and the murine endothelial cell line, SVEC4-10. Neovascularization was assayed by the tubulogenesis and scratch wound assays as well as quantitating the expression of various provasculogenic markers. Conditioned media, generated by culturing the murine mammary cancer cell line, TG1-1, in estrogen and ethanol, was observed to enhance tubule formation and migration of the murine endothelial cell line, SVEC4-10. In addition, the TG1-1 conditioned media enhanced the prosurvival MAP kinase-signaling pathway in SVEC4-10 cells. Presumably, the observed enhancement of both endothelial cell tubulogenesis and migration is in part attributed to provasculogenic proteins secreted by TG1-1 cells. In order to address this, Western blot analysis using TG1-1 cell lysates indicated an increase in VEGF, MMP-9, and eNOS expression in presence of alcohol and estrogen. As a whole, our data suggest that both estrogen and alcohol function to enhance expression of key provasculogenic molecules in breast cancer cells, which in turn modulates the morphologic, migratory, and survival properties of endothelial cells.

Citation Format: Natalie C. Cruz, Rachana Maniyar, Robert Suriano. Alcohol enhances estrogen-responsive breast cancer neovascularization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2059.
Abstract 2061: Genipin inhibits invasion and migration of colon cancer cells by suppression of HIF1α accumulation and VEGF expression
Authors: Lee Dae-Hee , Oh Sang-Choul , Lee Suk-young , Kim Hong Jun
Published: August 2018

Expression of hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) is known to play an important role in cancer progression in various cancer cell lines. Although genipin, a constituent of Gardenia fruit, has shown to have anti-tumor activity, its role in suppression of HIF-1 protein and downstream genes has yet to be clarified. We examined the effect of genipin on intracellular level of HIF-1α and extracellular level of VEGF proteins using colon cancer cell line HCT116. We observed genipin suppressed accumulation of HIF-1α under hypoxia in various cancer cell lines including HCT116. We found out suppressed accumulation of HIF-1α occurred through modulation of protein degradation. Genipin was also observed to suppress expression of VEGF and invasion of colon cancer cells through blocking of the ERK signaling pathway. Taken together, our results provide a new insight into the potential role of genipin in suppressing cancer progression of colon cancer cells.

Citation Format: Dae-Hee Lee, Sang-Choul Oh, Suk-young Lee, Hong Jun Kim. Genipin inhibits invasion and migration of colon cancer cells by suppression of HIF1α accumulation and VEGF expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2061.