bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2761: Development of synthetic guide RNA libraries for CRISPR-mediated transcriptional activation screening for gain-of-function studies
Authors: Vermeulen Annaleen , Strezoska Zaklina , Dickerson Sarah M. , Gross Maren M. , Chou Eldon T. , Maksimova Elena , Perkett Matthew R. , McClelland Shawn , Smith Anja V.
Published: August 2019

Functional gene analysis studies have been empowered by development of CRISPR-Cas9 gene knockout tools, however the CRISPR-Cas9 system has also been adapted for inhibition or activation of gene transcription. A nuclease-deactivated Cas9 (dCas9) can be fused to various effector domains to produce an RNA-guided transcription factor for either inhibition (CRISPRi) or activation (CRISPRa) of target genes. For overexpression studies, CRISPRa holds significant advantages over traditional vector-based gene expression, because genes are upregulated from their native promoter and endogenous genomic context. The majority of CRISPRa research performed to date has utilized single guide RNA (sgRNA) expressed from a DNA vector, primarily in the context of pooled lentiviral screening approaches. Here we describe the development of CRISPRa synthetic guide RNAs for the use in arrayed screening, so that we combine this next-generation transcriptional activation method with the ability to support more complex assays in a one-gene-per well format. Considerations for arrayed activation screens will be shown. The combination of gain-of-function from CRISPRa with loss-of-function using RNAi or canonical CRISPR-Cas9 for gene knockout allows for robust characterization of gene mechanisms and pathways.

Citation Format: Annaleen Vermeulen, Zaklina Strezoska, Sarah M. Dickerson, Maren M. Gross, Eldon T. Chou, Elena Maksimova, Matthew R. Perkett, Shawn McClelland, Anja V. Smith. Development of synthetic guide RNA libraries for CRISPR-mediated transcriptional activation screening for gain-of-function studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2761.
Abstract 2765: Exosomal miRNAs as circulating biomarkers for prediction of metastasis in stage II/III gastric cancer
Authors: Liu Xin , Chu Kent-Man
Published: August 2019

Background: Metastasis is the fatal character of cancer, leading to mortality of gastric cancer. In recent years, exosomes have been reported as extracellular vesicles secreted by cells. They contain various biological molecules including miRNAs from the original cells. Exosomal miRNAs are significant for pre-metastatic niche formation, as they can change the microenvironment and make it favorable for future metastasis. Therefore, the aim of this study was to identify dysregulated exosomal miRNAs as circulating biomarkers for prediction of metastasis of gastric cancer.

Materials and Methods: Pre-treatment serum samples of 36 metastatic patients and 55 non-metastatic patients with stage II/III gastric cancer were collected. Exosomes were extracted and validated by western blot, transmission electron microscope and nanoparticle tracking analysis. MiRCURY LNATM miRNA miRNome PCR Array (containing 752 miRNAs) was performed in three pairs of serum exosomes. Each pair samples were with the same stage, race, gender and similar age. These three pairs of samples represented stage II, IIIA or IIIB gastric cancer, respectively. A panel of commonly dysregulated exosomal miRNAs was released from the PCR Array. Expressions of exosomal miRNAs were validated in the remaining 33 metastatic and 52 non-metastatic patients by RT-qPCR, as well as in gastric cancer cell culture medium and cell lines.

Results: By comparing the expressions of exosomal miRNAs between the metastatic patients and matched non-metastatic ones, 13 upregulated and 6 downregulated miRNAs were released after normalization with miR-16-5p and miR-93-5p. Among these miRNAs, 7 of them were selected for further validation according to their fold changes, p-values and association with cancer development. MiR-379-5p and miR-410-3p were validated to be significantly upregulated in metastatic patients (P<0.01). Sensitivity, specificity and AUC were 67.31%, 69.70% and 0.6894 for miR-379-5p, and 65.38%, 66.67% and 0.6719 for miR-410-3p. Higher expression of serum exosomal miR-379-5p or miR-410-3p showed shorter overall survival of the patients (P<0.05). It was also found that miR-379-5p and miR-410-3p expressed significantly higher in gastric cancer cell culture medium comparing with gastric cancer cells.

Conclusions: Exosomal miRNAs are dysregulated in the serum of metastatic gastric cancer patients before they are clinically diagnosed. Upregulation of serum exosomal miR-379-5p and/or miR-410-3p are promising circulating biomarkers for prediction of metastasis in stage II/III gastric cancer.

Citation Format: Xin Liu, Kent-Man Chu. Exosomal miRNAs as circulating biomarkers for prediction of metastasis in stage II/III gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2765.
Abstract 2779: miR-142-3p as a regulator of RAC1 and a prognostic factor in melanoma
Authors: Cruz Adriana T. , Morata Júlia A. , Monteiro Ana C. , Ayub Ana Luísa P. , Soares Roseli S. , Fujita André , Andrade Fernando , Tron Victor , Jasiulionis Miriam G.
Published: August 2019

In order to identify miRNAs able to distinguish melanoma patients with good or poor prognosis, the expression profile of a panel of miRNAs evaluated in primary melanoma samples from 65 patients was compared to clinicopathological data from these patients (Breslow’s depth, mitotic counts, metastasis progression and survival). The miR-142-3p was identified as presenting at least 2-fold reduced expression in melanoma samples from patients with poor prognosis for 3 of 4 clinical parameters analyzed compared to melanoma samples from patients with good prognosis. The miR-142-3p expression was validated by RT-qPCR in primary human melanoma samples, confirming a significant lower expression in melanoma patients with poor prognosis compared to those with good prognosis. Searching for miR-142-3p mRNA targets in melanoma, the gene expression profile was analyzed by gene microarrays in a wild type and miR-142-3p overexpressing melanoma cell line. RAC1, ITGB8 e GNB2 were found as potential targets of this miRNA. Protein expression analyses in melanoma cell lines overexpressing the miR-142-3p reinforced the regulation of RAC1 by this miRNA. Functional assays showed that the overexpression of miR-142-3p leads to a reduced migratory ability compared to control cells, indicating its role in cell migration process. Finally, multivariate COX analyses demonstrated that high levels of RAC1 and reduced levels of miR-142-3p predict poor progression-free survival of melanoma patients, revealing these molecules as potential prognostic biomarkers for melanoma patients.

Supported by FAPESP, CNPQ and CAPES

Short title: miR-142-3p as a prognostic factor in melanoma

Note: This abstract was not presented at the meeting.

Citation Format: Adriana T. Cruz, Júlia A. Morata, Ana C. Monteiro, Ana Luísa P. Ayub, Roseli S. Soares, André Fujita, Fernando Andrade, Victor Tron, Miriam G. Jasiulionis. miR-142-3p as a regulator of RAC1 and a prognostic factor in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2779.
Abstract 2780: IL13RA2 is differentially regulated in papillary thyroid carcinoma versus follicular thyroid carcinoma
Authors: Chong Siao Ting , Kok Catherine Y. , Tan Khee Ming , Guan Shou Ping , Lai Siang Hui , Lim Cindy , Hu Jiancheng , Sturgis Charles , Eng Charis , Lam Paula Y. , Ngeow Joanne
Published: August 2019

The interleukin-13 receptor alpha2 (IL13RA2), which is known to overexpressed in glioblastoma multiforme, plays a role in various cellular processes such as cell migration that may contribute to tumor progression. Studies have attributed IL13RA2 to invasion and metastasis in cancers of the ovary, breast, and pancreas but the pathological role of IL13RA2 in thyroid cancer is still unclear. This study aims to evaluate the expression of IL13RA2 in thyroid carcinomas and examine the role of IL13RA2 in progression of papillary thyroid cancer (PTC). We performed IL13RA2 immunochemical staining on tissue microarrays of 137 thyroid carcinomas and observed that IL13RA2 expression was significantly correlated with advanced tumor stage (pT3 / pT4; p=0.001) and regional lymph node metastasis (pN1; p<0.001). Moreover, the staining scores of IL13RA2 were significantly higher in PTC compared to follicular and anaplastic subtypes (p<0.02) and correlated with advanced tumor stage amongst PTC samples (pT3 / pT4; p=0.028). This differential profile of IL13RA2 in PTC was further validated in thyroid cancer cell lines overexpressing IL13RA2 to assay the effects on cell proliferation, cell migration and epithelial-mesenchymal transition (EMT) using CCK-8, transwell migration assay, qRT-PCR and western blot analyses. Knockdown of IL13RA2 in the PTC subtype B-CPAP cell line showed significantly reduced cell viability, cell migration and EMT markers including N-cadherin, Vimentin and Snail. Exogenous overexpression of IL13RA2 in another PTC cell line, K1 increased cell migration and EMT although cell proliferation was not affected. In summary, we demonstrated that IL13RA2 is differentially regulated in PTC and is involved in cell migration by enhancing EMT. The underlying molecular mechanisms on how IL13RA2 drives progression of thyroid cancer remains to be further investigated.

Citation Format: Siao Ting Chong, Catherine Y. Kok, Khee Ming Tan, Shou Ping Guan, Siang Hui Lai, Cindy Lim, Jiancheng Hu, Charles Sturgis, Charis Eng, Paula Y. Lam, Joanne Ngeow. IL13RA2 is differentially regulated in papillary thyroid carcinoma versus follicular thyroid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2780.
Abstract 2788: The interplay between tumor-cell derived FoxO1 and macrophage in the tumor microenvironment of esophageal squamous cell carcinoma
Authors: Wang Ying , Guan Xinyuan , Kam Woon Ngar
Published: August 2019

Introduction: Esophageal squamous cell cancer (ESCC) is the predominant histologic type in China with high incidence and mortality. FOXO1 is considered as a tumor suppressor in several cancers, but its role in tumor immunology still remain elusive. A better understanding of how FOXO1 mediates the secretion of downstream chemokines, such as CCL20, and thus its ability in regulating macrophage function would be of crucial for further investigation.

Materials and Methods: Quantitative PCR and immunohistochemistry were used to study the expression of FoxO1 in clinical ESCC samples. The survival analysis was performed based on the result of tissue microarray and corresponding clinicopathological information. Stable foxo1-transfected cell lines were constructed in human ESCC cell lines, and ELISA, western blotting and Quantitative PCR were conducted to evaluate the expression of foxo1 and its downstream molecule CCL20. The subsets of macrophages were identified through Flow cytometry and qPCR.

Results: The expression of FOXO1 was significantly upregulated in tumorous tissue when comparing to adjacent normal tissue and predicted a poor prognosis in ESCC patients. Besides, the analysis of TCGA database indicated that the markers of M2 macrophage were upregulated in foxo1-high expressing tumor tissues when compared with foxo1-low expressing tumor tissues. In vitro, we have successfully differentiated monocytic cell line THP-1 into macrophage M0, M1 and M2. The M1 and M2 markers (CD80, CD163 and CD68) were measured by flow cytometry, while their major cytokines (IL6, TNF-α, CCL17, CCL18, IL1β and IL10) were detected by qPCR. The ELISA, western blot and qPCR results showed that the expression of CCL20 was upregulated in foxo1-high expressing tumor cells when compared to negative control. The migration assay showed that ESCC cells transfected with foxo1 could recruit more M2 macrophages when compared with foxo1-low expression ESCC cells and the effect was diminished upon blockade with anti-CCL20 neutralizing antibody in tumor cells. Besides, we found out that M2 macrophage could promote the migration and EMT of tumor cells by using M2 conditioned medium.

Conclusions: FoxO1 displays an important role in M2 macrophage migration through the modulation of CCL20. This increased CCL20 reported in ESCC may become specific molecular/cellular processes operative in the TME of ESCC patients.

Note: This abstract was not presented at the meeting.

Citation Format: Ying Wang, Xinyuan Guan, Woon Ngar Kam. The interplay between tumor-cell derived FoxO1 and macrophage in the tumor microenvironment of esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2788.
Abstract 279: c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer
Authors: Soleimani Milad , Bhattacharjee Ritvik , Giri Uma , Kaoud Tamer S. , Dalby Kevin N. , Eckhardt S. Gail , Berg Carla L. Van Den
Published: August 2019

Breast cancer is the most prevalent type of cancer among women. Triple negative breast cancer (TNBC) is a highly aggressive and heterogeneous subtype of breast cancer. It is the most challenging subtype to treat mainly due its aggressive nature and lack of targets such as HER2 (human epidermal growth factor 2), PR (progesterone receptor), and ER (estrogen receptor) found in other subtypes. We have shown that inhibiting both EGFR (epidermal growth factor receptor)/HER2 with lapatinib and JNK with JNK-IN-8 causes synergistic cell death in 11 out of 12 TNBC cell lines. EGFR is expressed in up to 90% tumors and JNK activation is linked to TNBC phenotype, metastasis, and stem cell character, among others. Synergism was achieved in the resistant, low-EGFR expressing cell line when a FGFR inhibitor (BGJ398) was used instead of lapatinib in combination with JNK-IN-8. Combination drug effects were accompanied by elevated oxidative stress. Suppression of oxidative stress restored cell viability in cells treated with the drug combination. JNK-IN-8 treatment alone or in combination with lapatinib induced massive cytoplasmic vacuolization with little apoptotic blebbing. The vacuoles were shown to be membrane-derived and acidic using Lucifer Yellow and Acridine Orange, respectively. Pre-treatment of cells with bafilomycin A1 (lysosome inhibitor), 5-(N-Ethyl-N-isopropyl) amiloride (macropinocytosis inhibitor), or N-acetylcysteine (antioxidant) reduced vacuolization. A similar effect was observed when cells were serum-starved. JNK1 knockout mouse mammary tumor cells, in comparison with JNK2 knockout, were resistant to drug synergism and displayed significantly less oxidative stress and vacuolization. Our studies demonstrated that concomitant blockade of JNK and individual tumor-appropriate targets induce synergistic cell death in TNBC cells in response to oxidative stress. Moreover, JNK blockade resulted in the formation of serum-dependent, acidic vacuoles that were reversible by lysosome or macropinocytosis inhibition. Loss-of-function studies indicated that JNK1 was the key isoform responsible for the drug response in TNBC cells. These findings suggest that JNK is involved in a novel cell death pathway and is a robust target in combination therapies against TNBC.

Citation Format: Milad Soleimani, Ritvik Bhattacharjee, Uma Giri, Tamer S. Kaoud, Kevin N. Dalby, S. Gail Eckhardt, Carla L. Van Den Berg. c-Jun N-terminal kinase is involved in an oxidative stress- and vacuole-associated cell death pathway in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 279.
Abstract 2809: The induction of CD8+ T lymphocytes and PD-L1+ immune cells by neoadjuvant chemoradiotherapy for rectal cancer
Authors: Fukuoka Eiji , Yamashita Kimihiro , Sugita Yutaka , Arimoto Akira , Nakamura Tetsu , Kakeji Yoshihiro
Published: August 2019

Background; The relationship between tumor immunological microenvironment and irradiation has been studied so far. Based on these findings, we examined a tumor microenvironment of rectal cancer performed by neoadjuvant chemoradiotherapy (NACRT) with immunohistochemical analysis of surgical specimens. But, for introduction to clinical settings, the analysis is not yet sufficient. So, in more detail, we investigate tumor infiltrating CD8+ T cells by irradiation in mouse CT26 colon tumor model.

Methods; Locally advanced rectal cancer were enrolled and 68 were eligible for this study. Immunohistochemical staining of programmed cell death- lignad1(PD-L1), CD8, and CD163 was performed on specimens of all cases. In a mouse model, CT26 tumor cells were inoculated in mouse, tumors are irradiated with a single dose of 10Gy after 14 days. We analyzed CD8+ T cells infiltration of both irradiated mouse and no treated mouse by flow cytometry.

Results; There were 44 cases in which NACRT was performed (NA group) and 24 cases in which surgery alone was performed (SA group). In the SA group, there was no significant change in the infiltration of PD-L1+immune cells (PD-L1+IC) nor CD8 +cells before and after treatment, but in the NA group, the infiltrations of them were significantly increased. In multivariate analysis, low infiltration of CD8 +cells were identified as poor prognostic factors in disease-free survival. Moreover, in our mouse model, elevated numbers of tumor-infiltrating CD8+ T cells between day11 and day18 after irradiation were observed in tumors of irradiated mice compared to no treated mouse. We observed that among CD8+ T cells, cells that coexpress PD-1 and Tim-3 comprise the major population in irradiated mouse. PD-1+Tim-3+ CD8+ T cells produced more INF-γ compared to that by PD-1+Tim-3-CD8+ T cells and PD-1-Tim-3- CD8+ T cells in irradiated mouse and no treated mouse. Among the three populations PD-1+Tim-3+ population contained the largest fraction of central memory (CD44hiCD62Llow) cells, but the lowest fraction of central memory (CD44hiCD62Lhi) cells.

Conclusion; The infiltration of PD-L1+IC and CD8 +cells was induced by NACRT. In the mouse model, we confirmed the kinetical and phenotypical changes of tumor-infiltrating CD8+ T cells.

Citation Format: Eiji Fukuoka, Kimihiro Yamashita, Yutaka Sugita, Akira Arimoto, Tetsu Nakamura, Yoshihiro Kakeji. The induction of CD8+ T lymphocytes and PD-L1+ immune cells by neoadjuvant chemoradiotherapy for rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2809.
Abstract 281: Sensitivity to splicing modulation of BCL2 family genes reveals cancer therapeutic strategies for splicing modulators
Authors: Aird Daniel , Teng Teng , Huang Chia-Ling , Pazolli Ermira , Banka Deepti , Cheung-Ong Kahlin , Eifert Cheryl , Furman Craig , Wu Jeremy , Seiler Michael , Buonamici Silvia , Fekkes Peter , Karr Craig , Palacino James , Park Eunice , Smith Peter , Yu Lihua , Mizui Yoshiharu , Warmuth Markus , Chicas Agustin , Corson Laura , Zhu Ping
Published: August 2019

Dysregulation of RNA splicing by spliceosome mutations or in cancer genes is increasingly recognized as a hallmark of cancer. Small molecule splicing modulators have been introduced into clinical trials to treat solid tumors or leukemia bearing recurrent spliceosome mutations. Nevertheless, further investigation of the molecular mechanisms that may enlighten therapeutic strategies for splicing modulators is highly desired. Here, using unbiased functional approaches, we report that the sensitivity to splicing modulation of the anti-apoptotic BCL2 family genes is a key mechanism underlying preferential cytotoxicity induced by the SF3b-targeting splicing modulator E7107. While BCL2A1, BCL2L2 and MCL1 are prone to splicing perturbation, BCL2L1 exhibits resistance to E7107-induced splicing modulation. Consequently, E7107 selectively induces apoptosis in BCL2A1-dependent melanoma cells and MCL1-dependent NSCLC cells. Furthermore, combination of BCLxL (BCL2L1-encoded) inhibitors and E7107 remarkably enhances cytotoxicity in cancer cells. These findings inform mechanism-based approaches to the future clinical development of splicing modulators in cancer treatment.

Citation Format: Daniel Aird, Teng Teng, Chia-Ling Huang, Ermira Pazolli, Deepti Banka, Kahlin Cheung-Ong, Cheryl Eifert, Craig Furman, Jeremy Wu, Michael Seiler, Silvia Buonamici, Peter Fekkes, Craig Karr, James Palacino, Eunice Park, Peter Smith, Lihua Yu, Yoshiharu Mizui, Markus Warmuth, Agustin Chicas, Laura Corson, Ping Zhu. Sensitivity to splicing modulation of BCL2 family genes reveals cancer therapeutic strategies for splicing modulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 281.
Abstract 2820: Macrophages recruited to and function to promote activation of cancer stem cells in response to chemotherapy
Authors: He Xi C. , Li Linheng
Published: August 2019

Failure of chemoradiotherapy for treating colorectal cancer (CRC) is often caused by the relapse of residual tumor cells that enrich cancer stem cells (CSCs). However, how CSCs are regulated by tumor microenvironment (TME), particularly myeloid derived cells (MDCs), remains unclear. Here, we used the APCMin/+ adenoma model to study the dynamic interaction between MDCs and CSCs in vivo following chemoradiotherapy. Using 3D scanning electronic microscopy, we observed that while proliferating tumor cells were dying following chemoradiotherapy, MDCs or its derivative tumor associated macrophages (TAMs) were recruited to the site where CSCs paired with necroptotic cells. TAMs functioned to promote activation of slow-cycling CSCs. Dissected with single cell RNA-seq, we observed that while proliferating tumor cells were declining following chemotherapy, an increase in MDCs was accompanied with a reduction of immune cells as well as an activation and short-term expansion of initially quiescent CSCs. Depletion of TAMs using a drugs or genetic means resulting in reduced CSCs and attenuated tumorigenesis. Mechanistically, we uncovered that TAM with secreted PGE2 activated Akt and Wnt signaling to promote activation of CSCs. Hence, targeting TAMs will benefit clinical treatment of CRC by reducing drug-resistant CSCs.

Note: This abstract was not presented at the meeting.

Citation Format: Xi C. He, Linheng Li. Macrophages recruited to and function to promote activation of cancer stem cells in response to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2820.
Abstract 2835: Breast tumor microbiota populations are modulated by chemotherapy and may be indicative of outcome
Authors: Chiba Akiko , Bawaneh Alaa , Velazquez Christine , Levine Edward , Cook Katherine L.
Published: August 2019

Breast tumors were identified to have their own microbiota population distinct from mammary gland tissue. Breast cancer patients that present in the clinic with larger tumors often undergo neo-adjuvant chemotherapy to reduce tumor burden before surgery. The purpose of our study was to evaluate whether chemotherapy can modulate the tumor microbiome and the potential impact of microbes in the development of distant metastases. Using snap-frozen aseptically collected breast tumor tissue from women that underwent neo-adjuvant chemotherapy or women with no prior therapy at time of surgery, we performed 16S rRNA sequencing to identify tumoral bacterial populations. We also stained breast tumor microarrays (normal breast tissue, primary breast tumor, and lymph node metastases) to confirm presence of identified microbiota. Our data indicates that chemotherapy administration significantly increased breast tumor Pseudomonas and reduced the proportional abundance of Streptococcus. Primary breast tumors from patients that developed distant metastases later on in life (regardless of therapy) displayed increased tumoral abundance of Acinetobacter, Brevundimonas, and Staphylococcus. Stratifying patients based upon BMI indicates that obesity modulates breast tumor microbiota populations and needs to be considered in analyses. Furthermore, we confirm presence of Pseudomonas in breast tumor tissue by immunohistochemical staining. Our results indicate breast tumor microbiota populations can be modified by chemotherapy and specific microbes correlate with tumor recurrence. Further studies with a larger patient cohort may provide greater insights into the role of microbiota in therapeutic outcome and for the development of novel bacterial biomarkers that could predict distant metastases.

Citation Format: Akiko Chiba, Alaa Bawaneh, Christine Velazquez, Edward Levine, Katherine L. Cook. Breast tumor microbiota populations are modulated by chemotherapy and may be indicative of outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2835.
Abstract 2847: Tenascin C expression in neural microenvironment enhances perineural invasion and is associated with locoregional recurrence-related poor prognosis in pancreatic ductal adenocarcinoma
Authors: Furuhashi Satoru , Sakaguchi Takanori , Murakami Tomohiro , Fukushima Mayu , Morita Yoshifumi , Ikegami Koji , Kikuchi Hirotoshi , Baba Megumi , Setou Mitsutoshi , Takeuchi Hiyoya
Published: August 2019

Background: Perineural invasion (PNI) is commonly seen in pancreatic ductal adenocarcinoma (PDAC) and worsens the postoperative prognosis. However, the associated mechanisms in cancer progression remain unclear. Tenascin C (TNC), an extracellular matrix glycoprotein, is abundant in cancer stroma and modulates tumor progression. The aim of this study was to evaluate the functional roles of TNC in the neural microenvironment of PDAC.

Methods: We immunohistochemically examined TNC expression in 78 resected PDAC specimens. TNC staining in perineural sites at the invasive front was classified as low or high intensity, by comparison with adjacent non-cancerous tissues in the same section. The relationships between TNC expression and clinicopathological features were retrospectively analyzed. Furthermore, interactions between cancer cells and nerves after supplementation with TNC were investigated when co-culturing a PDAC cell line with a mouse dorsal root ganglion (DRG) in vitro.

Results: High perineural TNC expression at the invasive front, seen in 30 (38%) patients, was associated with PNI (p = 0.006), pathological T stage ≥ 3 (p = 0.01), and postoperative locoregional recurrence (p = 0.002). It was independently associated with postoperative, poor recurrence-free survival in multivariate analysis (p = 0.045). In the in vitro co-culture model, supplementation with TNC significantly enhanced both neurotropism of PDAC cells and tumor tropism of a DRG. On the other hand, when PDAC cells and a DRG were cultured separately, TNC did not affect cancer cell proliferation or neural outgrowth.

Conclusions: Strong perineural TNC expression was a prognostic indicator of locoregional recurrence-related poor prognosis. The neurotropism of PDAC induced by TNC suggests that TNC could serve as a therapeutic target for PDAC.

Citation Format: Satoru Furuhashi, Takanori Sakaguchi, Tomohiro Murakami, Mayu Fukushima, Yoshifumi Morita, Koji Ikegami, Hirotoshi Kikuchi, Megumi Baba, Mitsutoshi Setou, Hiyoya Takeuchi. Tenascin C expression in neural microenvironment enhances perineural invasion and is associated with locoregional recurrence-related poor prognosis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2847.
Abstract 2850: Characterization of cadherin 6 as a novel therapeutic target in metastatic ovarian carcinoma
Authors: Burdiel Miranda , Bartolomé Ruben , Jaén Marta , Casal Ignacio
Published: August 2019

Cadherin 6 (CDH6) is a type II cadherin containing a RGD motif in a similar way to LI-cadherin (CDH17) and VE-cadherin (CDH5), which are able to activate α2β1 integrin signalling to promote lung and liver metastasis. CDH6 gene is frequently overexpressed in late stages of some types of ovarian carcinoma, clear cell renal carcinoma, renal papillary carcinoma and thyroid cancers. Therefore, as reported for CDH17 and CDH5, we hypothesized that CDH6 might be a potential therapeutic target for metastatic ovarian cancer. Indeed, an “in silico” analysis revealed a significant association between CDH6 expression, tumour stage and poor survival in renal and ovarian cancer patients.

We confirmed a high CDH6 expression in ovarian cancer cell lines, although with significant differences in cell surface availability, as determined by flow cytometry. In addition, we noticed a variable α2β1 integrin expression in the same cell lines. Indeed, CDH6 RGD peptide caused β1 integrin activation in OVCAR3 cells but not in SKOV-3, suggesting the existence of alternative mechanisms. In this regard, as CDH6 has been reported to be a αIIbβ3 integrin ligand in platelets, we investigated and confirmed the expression of αIIbβ3 integrin in SKOV3 but not in OVCAR3 cells. Remarkably, CDH6 was able to activate αIIbβ3 integrin in SKOV3 cells. Moreover, silencing of CDH6 or β3 integrin triggered a significant loss of prometastatic traits in ovarian cells, such as cell adhesion, migration, invasion and proliferation. Signalling pathway analysis demonstrated that β3 integrin activation was triggering the phosphorylation of AKT and SRC, but not FAK, promoting cell invasion. Finally, treatment with monoclonal antibodies against the cadherin RGD motif inhibited the metastatic behaviour in ovarian cancer cell lines “in vitro”. Experiments with metastatic mouse models to confirm the inhibitory capacity of cadherin RGD-specific monoclonal antibodies are in progress.

In summary, these findings suggest that CDH6 RGD motif could activate α2β1 or αIIbβ3 integrins, according to the cell context, and be targeted to avoid metastatic dissemination and cancer progression in those ovarian tumours overexpressing CDH6.

Citation Format: Miranda Burdiel, Ruben Bartolomé, Marta Jaén, Ignacio Casal. Characterization of cadherin 6 as a novel therapeutic target in metastatic ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2850.
Abstract 2851: The effects of nidogen-1 on proliferation and migration in claudin-low mammary tumor cells
Authors: Jagroop Rebecca , Moorehead Roger
Published: August 2019

Breast cancer is the most common type of cancer among women, with one subset of the triple-negative subtype, claudin-low, known to be very aggressive and metastatic. For invasion and metastasis to occur, cancer cells must cross basement membranes, which contain structural proteins such as laminin and collagen IV and linking proteins such as perlecan and nidogen, to colonize distant tissues. Nidogen is a glycoprotein that makes up 2-3% of basement membranes and has two types: nidogen-1 (NID1) and nidogen-2 (NID2). There have been limited studies on NID1 in cancer, with results demonstrating decreased invasiveness and metastatic capabilities in Nid1 silenced cells of various cancer types. In previous work, a murine cell line representative of the claudin-low subtype, known as RJ423, was developed; it demonstrated a 5000-fold increase in Nid1 expression compared to the luminal subtypes. To test whether high Nid1 expression contributes to the aggressive, metastatic nature of claudin-low tumors, Nid1 levels were knocked down in RJ423 cells and proliferation and migratory capabilities were assessed. Phospho-histone H3 immunofluorescence demonstrated that suppressing NID1 reduced RJ423 cell proliferation significantly. Also, proliferation was explored using flow cytometry to obtain cell cycle profiles, and it was observed that there was a reduction in the G2 phase of NID1-suppressed cells. Additionally, apoptosis was assessed through flow cytometry to determine annexin V levels, but suppression of NID1 levels did not alter apoptosis. Furthermore, invasion assays demonstrated a reduction in migration through collagen IV-coated wells using NID1-suppressed cells. Knockdown of Nid1 expression in RJ423 cells reduced the level of NID1 in conditioned media, and preliminary studies suggest that the levels of NID1 in conditioned media can influence tumor cell growth. These findings suggest that NID1 could be targeted to lessen the metastatic nature of claudin-low breast cancer.

Citation Format: Rebecca Jagroop, Roger Moorehead. The effects of nidogen-1 on proliferation and migration in claudin-low mammary tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2851.
Abstract 2855: Oleic acid-induced ANGPTL4 facilitates metastasis of human colorectal cancer via up-regulation of NOX4 expression
Authors: Shen Chih-Jie , Liao Yu-Han , Tsai Jhih-Peng , Hung Liang-Yi , Chang Wen-Chang , Chen Ben-Kuen
Published: August 2019

Colorectal cancer (CRC) is highly associated with metabolic diseases, such as obesity and diabetes. Elevated free fatty acids (FFAs) in cancer patients with metabolic disorders may be associated with cancer progression. In addition, increased levels of reactive oxygen species (ROS) are also observed in dyslipidemia which induces expression of NADPH oxidase 4 (NOX4) by FFAs in aorta, kidney, and white adipocyte in physiology condition. Although the expression of NOX4 has been also up-regulated in various cancer cell types, the correlation between up-regulation of NOX4 and dyslipidemia-regulated CRC metastasis remains unclear. Here, we found that oleic acid (OA) induced-angiopoietin-like 4 (ANGPTL4) expression was through the activation of PPARs pathways, resulting in promoting the metastasis of CRC. The recombinant protein of human ANGPTL4 rescued the OA-induced invasive ability in ANGPTL4 knockdown cells. It is worthy to note that the knockdown of ANGPTL4 not only significantly inhibited OA-induced NOX4 expression, but also reduced ROS production. In addition, the depletion of ROS by using NAC or knockdown of NOX4 inhibited OA-induced extravasation of CRC cells in vivo. OA-induced MMP1 and MMP9 expressions were also dependent on the expression of ANGPTL4 and NOX4 in CRC cells. The transcriptional activation of NOX4 gene by OA-induced ANGPTL4 was regulated by activation of c-Jun and dependent on AP-1 site of NOX4 promoter. These results reveal that OA-promoted CRC metastasis was through the activation of ANGPTL4/NOX4 axis, suggesting that ANGPTL4 and NOX4 may be potential therapeutic targets and diagnostic markers to improving outcomes for patients with CRC.

Citation Format: Chih-Jie Shen, Yu-Han Liao, Jhih-Peng Tsai, Liang-Yi Hung, Wen-Chang Chang, Ben-Kuen Chen. Oleic acid-induced ANGPTL4 facilitates metastasis of human colorectal cancer via up-regulation of NOX4 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2855.
Abstract 2857: Vimentin is a novel PKC-ι target facilitating migration and invasion in melanoma
Authors: Ratnayake Wishrawana Sarathi , Breedy Sloan , Apostolatos Christopher , Hill Robert , Dennison Clare , Acevedo-Duncan Mildred
Published: August 2019

Vimentin plays an important role in gaining rear-to-front polarity for mesenchymal cells, making it a hallmark of cancer metastasis. Epithelial-mesenchymal transition (EMT) is a vital process in tumor progression that converts stationary epithelial cells into motile mesenchymal phenotype by downregulating E-cadherin and upregulating Vimentin expression. Molecular dynamics of Vimentin intermediate filaments (VIFs) play several key roles in cancer metastasis. Vimentin is activated via phosphorylation through various kinases. Recently, we reported that Protein Kinase C-iota (PKC-ι), activates Vimentin in melanoma while inducing EMT. We also reported the effects of PKC-ι inhibition on mitigating EMT in melanoma based on PKC- ι specific inhibitors {Ratnayake, W.S., et al. Int. J. Oncol. 51(5), (2017), 1370-1382 https://doi.org/10.3892/ijo.2017.4131 and Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. This novel finding is very important with respect to cancer progression since PKC-ι is a well-established oncogene in various tumor types. Here, we analyzed the VIF dynamic changes upon PKC-ι inhibition using specific inhibitors targeting multiple activation sites on Vimentin. In addition to siRNA knockdown of expression of PKC-ι, we also report the effects of siRNA knockdown of two transcription factors, Paired related homeobox1 (PRRX1) and Zinc finger protein SNAI1 (SNAIL1) on the expressions of Vimentin, PKC-ι and E-cadherin. Herein we show that inhibition of PKC-ι downregulate the transcriptional activities of PRRX1 and SNAIL1, which was demonstrated with diminished levels of Vimentin and elevated levels of E-cadherin. Western blot, confocal immunofluorescence and immune-gold electron microscopic results suggested that PKC-ι targets multiple activation sites (S33, S39 and S56) on Vimentin without having an effect on S6 and S71 phosphorylation sites. Results also suggest that PKC-ι inhibition leads to diminution of activities of PRRX1 and SNAIL1 thereby further downregulation of Vimentin while upregulating E-cadherin in order to continue retardation of EMT in-vitro. Overall results indicate that PKC-ι is essential for VIF dynamics regulation during the metastasis in-vitro and therefore can be used as a novel biomarker to assess and mitigate melanoma metastasis.

Citation Format: Wishrawana Sarathi Ratnayake, Sloan Breedy, Christopher Apostolatos, Robert Hill, Clare Dennison, Mildred Acevedo-Duncan. Vimentin is a novel PKC-ι target facilitating migration and invasion in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2857.
Abstract 2861: ACKR3 promotes cell metastasis via activating TGF-beta 1/Smad signaling in head and neck squamous cell carcinoma
Authors: Kim Nayoung , Kim Solbi , Joo Mina , Jeon Heung Jin , Lee Myung-Won , Ryu Hyewon , Lee Hyo Jin
Published: August 2019

Background: The atypical chemokine receptor, ACKR3, has been shown to play as a tumor promoter, which is upregulated in various types of cancer. However, the biological role of ACKR3 and its underlying mechanism in head and neck squamous cell carcinoma (HNSCC) is still unknown. In this study, we investigated the functional role of ACKR3 and the underlying molecular mechanism of tumor progression in HNSCC.

Methods: We examined the ACKR3 expression and its association with clinicopathological characteristics in 103 cases of HNSCC by immunohistochemical staining. The biological roles of ACKR3- and ACKR3-induced downstream signaling were investigated in HNSCC cell through ACKR3 overexpression and knockdown of ACKR3 in vitro and in vivo.

Results: We detected differential expression of ACKR3 in HNSCC cells and tissues. High ACKR3 expression was significantly associated with invasion depth of tumor (T status; P = 0.007), lymph node involvement (N status; P = 0.004), and higher stage III/IV (P = 0.02). Overexpression of ACKR3 dramatically enhanced cell migration and invasion in HNSCC cells in vitro, and promoted lymph node metastasis in vivo. ACKR3 overexpression also induced the epithelial-mesenchymal transition. Vimentin, Slug, and Twist were increased but E-cadherin and Ep-CAM were decreased by ACKR3 expression. Upregulation of TGF-β1 was observed in ACKR3 overexpression, and TGF-β1/Smad signaling activation and Akt phosphorylation were induced in HNSCC cells with ACKR3 overexpression. Treatment with a PI3K inhibitor reduced Slug and Twist levels while suppression of Smad2/3 signaling by siRNA reduced Akt phosphorylation, as well as Slug and Twist. Furthermore, inhibition of Smad2/3 decreased tumor cell migration and invasion in HNSCC. ACKR3 knockdown using siRNA in HNSCC cells reduced the secretion of TGF-β1 and also recovered the cell migratory and invasive behavior of HNSCC cells.

Conclusions: ACKR3 expression was associated with an aggressive tumor behavior in HNSCC. ACKR3 contributed to migration and invasion of HNSCC cell through the TGF-β1/Smad signaling axis and epithelial-mesenchymal transition in vitro, and was involved in lymph node metastasis in vivo, suggesting that ACKR3 plays an important role during HNSCC progression and metastasis.

Citation Format: Nayoung Kim, Solbi Kim, Mina Joo, Heung Jin Jeon, Myung-Won Lee, Hyewon Ryu, Hyo Jin Lee. ACKR3 promotes cell metastasis via activating TGF-beta 1/Smad signaling in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2861.
Abstract 2863: Long non-coding RNA LINC00152 plays an oncogenic role via targeting MET, STAT3 signaling in esophagus adenocarcinoma
Authors: Xiao Matthew , Wang Zhuwen , Lin Jules , Beer David G. , Chang Andrew C. , Chen Guoan
Published: August 2019

Esophageal cancer remains a threat to public health with an increasing incidence and low survival rate world wide. Long non-coding RNA, LINC00152 has been linked to several human cancers and promotes cell proliferation in lung, gastric, hepatocellular, colorectal, and clear cell renal carcinoma. The expression of LINC00152 and its roles in esophagus adenocarcinomas (EAC), however, are unknown. The objective of this study is to investigate the role of LINC00152 in EAC. We used the esophageal cancer cell lines Flo-1, OE19, and OE33 for in vitro study. siRNA of specific to LINC00152 was used to knockdown LINC00152 followed by cell proliferation, colony formation, invasion and migration assays. Real-time PCR assay was used for detecting mRNA expression and Western blot for examining altered proteins expression affected by LINC00152 knockdown. Data analysis were performed using excel and Prism. The difference of variables was analyzed using the Student’s T-test. We found that LINC00152 was increased in EAC as compared to Barrett, low-grad displasia or high-grad dysplasia. The LINC00152 expression was decreased more than 90% after LINC00152 knockdown with siRNA on Flo, OE19 and OE33 cells measured by RT-PCR. Cell proliferation, colony and invasion were decreased significantly after LINC00152 siRNA transfection on these EAC cell lines. Apoptosis was also induced. Mechanistic study indicated that MET protein was significantly decreased in OE33 cells after LINC00152 knockdown, while STAT3 decreased in Flo cells. P21, p27 and Myc was decreased in all 3 cell lines. In this study we found that LINC00152 knockdown could inhibit cell proliferation, colony formation, cell migration and invasion, as well as trigger apoptosis via targeting multiple oncogenic signaling. We also found LINC00152 was increased in EAC tumors. Our study indicates that LINC00152 plays an important role in EAC and is potentially a diagnostic marker. Further characterization of LINC00152 as a novel therapeutic target of EAC is warranted.

Citation Format: Matthew Xiao, Zhuwen Wang, Jules Lin, David G. Beer, Andrew C. Chang, Guoan Chen. Long non-coding RNA LINC00152 plays an oncogenic role via targeting MET, STAT3 signaling in esophagus adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2863.
Abstract 2913: Serpinb3a-/- mice are radioresistant
Authors: Thermozier Stephanie , Epperly Michael , Franicola Darcy , Zhang Xichen , Fisher Renee , Shields Donna , Wang Hong , Willis John , Luke Cliff , Silverman Gary , Greenberger Joel
Published: August 2019

Serpins are a superfamily of serine protease inhibitors that regulate proteolytic pathways by utilizing a conformational change to bind to and inhibit their target peptidases. Currently 36 human serpins have been identified, but the biological roles of most are not fully understood (Silverman et al., J Biol Chem. 285(32):24299-305, 2010). Serpinb3a (the mouse orthologue of human SERPINB3 and -B4) is an intracellular serpin and inhibits both serine and cysteine peptidases invitro. We now report radioresistance of Serpinb3a-/- mice and their hematopoietic progenitor cells. Serpinb3a-/- and control Balb/c mice were irradiated to a dose of 8.0 Gy total body irradiation and followed for the development of the hematopoietic syndrome. Long Term Bone Marrow Cultures (LTBMCs) were established from the bone marrow flushed from the femurs and tibias of Serpinb3a-/- and Balb/c mice. Irradiation survival curves were performed on bone marrow stromal cell lines established from the LTBMCs and on fresh bone marrow isolated from the femurs of sacrificed Serpinb3a-/- and Balb/c mice. Colony forming unit granulocyte-macrophage (CFU-GM) colonies of 50 cells or greater were counted. The data from 3 experiments were analyzed using linear quadratic and single-hit, multi-target models. Serpinb3a-/- mice showed increased survival following 8.0 Gy TBI compared to Balb/c mice (p < 0.0001). CFU-GM from Serpinb3a-/- were radioresistant compared to Balb/c marrow (ň = 4.9 ± 1.4 and 1.4 ± 0.1, p = 0.0451, respectively). Serpinb3a-/- bone marrow stromal cells were radioresistant with an increased Do (1.97 + 0.11 Gy) compared to bone marrow stromal cells from Balb/c mice (Do = 1.45 + 0.03 Gy, p = 0.0113). Loss of Serpinb3a provides a radiation survival advantage, increases radioresistance of both hematopoietic progenitor cells and marrow stromal cells, and may lead to identification of new targets for design of radiation mitigator drugs.

This research was supported by grant NIAID/NIH U19-A168021.

Citation Format: Stephanie Thermozier, Michael Epperly, Darcy Franicola, Xichen Zhang, Renee Fisher, Donna Shields, Hong Wang, John Willis, Cliff Luke, Gary Silverman, Joel Greenberger. Serpinb3a-/- mice are radioresistant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2913.