bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4009: Biotinylated estrogens a novel tool for early detection of adduct in ovarian cancer and their carcinogenic effects in cultured human cells
Authors: Tripathi Kaushlendra
Published: August 2018

In humans, endogenous estrogens such as Estrone (E1) and 17L-estradiol (E2) regulate many physiological processes including reproduction, primarily in females, and control the growth of certain responsive cells. However, unbalanced estrogen metabolism has been linked to development of several malignant diseases including breast, endometrial and ovarian cancer and has been identified as a risk factor for these cancers. Elevated estrogens exert their carcinogenic effects by at least three different mechanisms; they promote cell proliferation by transcriptional regulation of estrogen responsive genes, cause generation of reactive oxygen species and other free radicals, and directly react with DNA and form potentially mutagenic adducts. The metabolic breakdown of estogens is regulated by a series of reactions mediated by hepatic and peripheral enzymes that balances the induction and reduction of cellular stress. An imbalance in this enzymes and processes can lead to highly reactive catechol estrogen metabolites that can react with DNA, form carcinogenic DAN-adducts and lesions. Although several studies established a connection between estrogen-induced DNA damage and carcinogenesis, the underlying molecular mechanisms have been difficult to study because of the technical challenges in detecting and analyzing the variety of different DNA lesions that are formed by estrogen compounds. Moreover, detection and analysis of these adducts are important to directly monitor estrogen metabolites induced cellular responses in the cells. Towards this, we developed a novel method using biotinylated-estrogens that allows immunodetection of estrogen-induced DNA adducts by Slot-blot and single-cell molecular combing and proximity ligation assays. Using these modified estrogens we first time quantitatively detected these adducts on DNA by immune Slot-blot techniques and on DNA fibers. Furthermore, similar to other environmental carcinogens estrogens activates replication associated DNA damage responses and induces chromosomal instability. Thus, for the first time our studies demonstrate that biotin-labeled estrogens could be a powerful tool to detect estrogen adducts and to probe associated DNA damage responses and cellular responses.

Citation Format: Kaushlendra Tripathi. Biotinylated estrogens a novel tool for early detection of adduct in ovarian cancer and their carcinogenic effects in cultured human 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 4009.
Abstract 4023: Gap junctions contribute to anchorage-independent clustering of breast cancer cells
Authors: Gava Fabien , Rigal Lise , Mondesert Odile , Pesce Elise , Ducommun Bernard , Lobjois Valérie
Published: August 2018

Purpose: Cancer cell cluster formation is a key process involved during primary tumor growth, metastatic cells dissemination and secondary tumor evolution. We previously reported that cell-cell adhesion proteins such as E-Cadherin and desmosomal proteins are involved in cell aggregation independently of cell migration or matrix adhesion (Saias et al. 2015). Here we investigate the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells.

Experimental Design: Live cell imaging and image processing was used to evaluate MCF7 clustering in the presence of pharmacological inhibitors and to perform a LOPAC® small bioactive compound library screening. Flow cytometry was performed to monitor calcein transfer assay and to evaluate GJIC.

Results: We first demonstrate that functional GJIC are established in the early phase of aggregation. We next show that pharmacological inhibition of GJIC using Tonabersat and Meclofenamate both reduces calcein transfer and delays clustering of MCF7 cells. We found that treatment with Latrunculin A, an actin cytoskeleton-disrupting agent, inhibits MCF7 clustering and impairs calcein transfer. In a screen for inhibitors of cell aggregation using a small-compound chemical library we identified Brefeldin A, an inhibitor of vesicular trafficking, and showed that it also impairs calcein transfer.

Conclusion: Our results demonstrate that GJIC are involved at the earliest stages of the anchorage-independent clustering process. They shed new light on the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.

References:

Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, Yu M, Pely A, Engstrom A, Zhu H et al: Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 2014, 158(5):1110-1122.

Saias L, Gomes A, Cazales M, Ducommun B, Lobjois V: Cell-Cell adhesion and cytoskeleton tension are key but opposing regulators of tumor cell aggregation. Cancer Research 2015, 75(12):1-8.

Gava, F., Rigal, L ., Mondesert, O., Pesce, E., Ducommun, B. and Lobjois, V. Gap junctions contribute to anchorage-independent clustering of breast cancer cells (Submitted).

Citation Format: Fabien Gava, Lise Rigal, Odile Mondesert, Elise Pesce, Bernard Ducommun, Valérie Lobjois. Gap junctions contribute to anchorage-independent clustering of 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 4023.
Abstract 4024: TRAF2 Ser-11 phosphorylation confers resistance to AKT inhibitors in breast cancer cells
Authors: Habelhah Hasem , Zhang Laiqun
Published: August 2018

The PI3K-AKT pathway is the most frequently aberrantly activated pathway in breast cancer (BC), with alterations in genes encoding the pathway components (e.g. RTKs, HER2, PTEN, PIK3C and AKT) occurring in ~80% of cases. However, inhibition of this pathway is either not effective or often results in development of resistance in BC. TRAF2 is a key effector protein downstream of the TNF receptor superfamily, and activates NF-κB to promote BC cell survival. We mapped two phosphorylation sites (Ser-11 and Ser-274) in TRAF2, and identified TBK1/IKKϵ and AKT as the kinases that directly phosphorylate TRAF2 at Ser-11 and Ser-274, respectively. TBK1 and IKKϵ are serine/threonine kinases activated by inflammatory stimuli and the oncogenic RTK-Ras-RALB pathway, and play a critical role in BC transformation and survival. IKKϵ and TRAF2 are overexpressed in a significant portion of BC, and their overexpression is associated with poor prognosis in patients with invasive breast carcinoma. We found that inhibition of AKT leads to increased TBK1 activation and TRAF2 Ser-11 phosphorylation in triple negative BC (TNBC) cells. Importantly, combination of AKT and TBK1/IKKϵ inhibitors synergistically induced apoptosis in all TNBC cell lines tested in vitro, suggesting that inhibition of PI3K-AKT derepresses the pro-survival TBK1-TRAF2 pathway, which in turn confers resistance to AKT inhibition in TNBC. We believe that any positive outcomes from our ongoing xenograft experiments will have an immediate translational impact in TNBC treatment.

Citation Format: Hasem Habelhah, Laiqun Zhang. TRAF2 Ser-11 phosphorylation confers resistance to AKT inhibitors in 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 4024.
Abstract 4025: An old foe with a new function: JAM-A regulates expression of EphB4 and inhibitor of apoptosis proteins in breast cancer
Authors: Vellanki Sri HariKrishna , Cruz Rodrigo G. , Richards Cathy E. , Brennan Kieran , Hopkins Ann M.
Published: August 2018

Over-expression of Junctional Adhesion Molecule-A (JAM-A) in breast tumors has been linked with increased risk of metastasis. Since JAM-A is co-expressed with the receptor tyrosine kinase (RTK) HER2 and regulates HER2 protein expression, we hypothesized that JAM-A regulates the expression and function of other RTKs. Using a joint RT-PCR array/western blot approach in three breast cell lines with modified JAM-A expression, we identified EphB4 as a downstream target of JAM-A. Interestingly, EphB4 gene silencing also cross-regulated the protein expression of JAM-A. Immunofluorescence analysis revealed colocalisation of JAM-A and EphB4 at the cell membrane. Since JAM-A contains a PDZ-binding motif and EphB4 contains a PDZ domain, we speculated that this might underlie the basis of an interaction. Accordingly, overexpression of a JAM-A mutant lacking the PDZ domain failed to upregulate EphB4 expression to the same extent as wild type JAM-A. Next we investigated the signalling events downstream of JAM-A and EphB4 and found downregulation of the tight junction protein ZO-1 in breast cells silenced for JAM-A or EphB4. Using a bioinformatics approach, we identified the transcription factor c-FOS as a potential regulator of JAM-A and EphB4 expression. In JAM-A- or EphB4-silenced cells, c-FOS expression was not localized in the nucleus (relative to cells transfected with negative control siRNAs). Furthermore, ERK5 phosphorylation was inhibited in JAM-A- or EphB4-silenced cells, suggesting a mechanism for reduced c-FOS phosphorylation on Serine-32 and its consequent exclusion from the nucleus. In the context of a proposed block on c-FOS transcriptional activity, we examined the genes affected and show for the first time downregulation of inhibitor of apoptosis proteins (IAPs) in JAM-A- or EphB4-silenced cells. In conclusion, our data are consistent with a model whereby JAM-A regulates EphB4 expression in a PDZ-dependent manner and influences apoptosis via a pathway involving ERK5 and c-FOS. Our data suggest that the pharmacological antagonism of JAM-A in JAM-overexpressing breast cancers merits consideration by virtue of its predicted ability to downregulate EphB4 expression and remove a blockade on apoptosis.

Citation Format: Sri HariKrishna Vellanki, Rodrigo G. Cruz, Cathy E. Richards, Kieran Brennan, Ann M. Hopkins. An old foe with a new function: JAM-A regulates expression of EphB4 and inhibitor of apoptosis proteins in breast 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 4025.
Abstract 4037: Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial cells
Authors: Trevizo-Barresi Brenda G. , Welsh JoEllen
Published: August 2018

Evidence suggests that growth of cancer stem cells (CSCs) drives intratumor heterogeneity, a major clinical problem that results in drug resistance, recurrence of tumors, and metastatic progression of breast cancer. Therapeutic strategies to eliminate CSCs require identification of signaling pathways that facilitate the emergence and/or expansion of CSCs. These signaling pathways are implicated in altering cell behavior, as well as promoting the expression of genes involved in epithelial-mesenchymal transition (EMT), cell migration and invasion. Previous work demonstrated that a cooperative STAT3/SMAD3 gene transcription program increased the expression of Hyaluronan Synthase 2 (HAS2), an enzyme that produces Hyaluronic Acid (HA) which serves as a CD44 ligand and facilitates EMT. We hypothesize that STAT3 is a critical regulator of HA synthesis and signaling which enhances EMT and stemness of human mammary epithelial cells, driving tumor progression. We tested this hypothesis in a TGFβ triggered model of EMT. Immortalized human mammary epithelial cells (HMLE) expressing either a control vector (pWZL-GFP) or a vector containing TGFβ1 (pWZL-TGFβ1-GFP) were characterized with respect to STAT3 signaling, HA synthesis, and mammosphere formation. Our data revealed that HMLE-pWZL-TGFβ1-GFP cells have significantly higher expression of active STAT3 (Y705) compared to control cells, but total STAT3 gene and protein expression were similar in the two cell lines. HMLE-pWZL-TGFβ1-GFP cells exhibited a significant increase in expression of HAS2 and HAS2-AS1, a long non-coding RNA that enhances the transcription of HAS2. Preliminary results show that overexpression of TGFβ1 increased HA secretion in media compared to control cells. HMLE-pWZL-TGFβ1-GFP cells demonstrated significantly higher mammosphere forming ability compared to control cells, suggesting that TGFβ increases the percentage of stem cells in the population. Initial results demonstrated changes in mammosphere size and morphology of HMLE-pWZL-TGFβ1-GFP cells treated with the HA synthesis inhibitor 4-Methylumbelliferone (4MU). Together, these results suggest STAT3 and TGFβ1 co-regulate HAS2 expression and HA production in a model of EMT supporting the use of the HMLE-pWZL cell series as a promising model to study STAT3 regulation of the HA-CD44 signaling pathway.

Citation Format: Brenda G. Trevizo-Barresi, JoEllen Welsh. Interactions between STAT3 and TGFβ in the regulation of hyaluronic acid synthesis and signaling in immortalized human mammary epithelial 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 4037.
Abstract 4039: PAX8 increases migration and metastasis of high grade serous ovarian cancer through upregulation of Rho GTPases
Authors: Hardy Laura , Pergande Melissa , Hernandez Angel , Cologna Stephanie , Burdette Joanna
Published: August 2018

High grade serous ovarian cancer, the most lethal subtype of ovarian cancer, can originate in either the fallopian tube epithelium (FTE) or ovarian surface epithelium (OSE). PAX8 is a lineage specific transcription factor that is ubiquitously expressed in HGSOC. We have shown that knockdown of PAX8 using shRNA in multiple ovarian tumor cells lines leads to apoptosis, suggesting that PAX8 plays an essential role in cancer survival. In this study, we used CRISPR technology to delete PAX8 from the OVCAR8 cell line. PAX8 deletion led to a decrease in migration and invasion in vitro and an increase in survival and a reduction in tumor volume in vivo. Previous work using RNA-sequencing and ChIP-sequencing identified cell adhesion as a top differentially expressed gene between malignant ovarian cancer and benign fallopian tube cell lines. We performed quantitative proteomic analysis of the OVCAR8-PAX8-/- cell line to define PAX8 altered proteins. We also performed quantitative proteomics and transcriptomic analyses on a previously generated murine OSE cell line with forced PAX8 expression (MOSE-PAX8). These analyses identified several genes that contribute to an increase in migration and EMT. Specifically, our data indicates PAX8 upregulates key drivers involved in altering cell morphology including the GTPases: RhoA, Cdc42, and Ras. Inhibition of RhoA led to a greater decrease in migration for both OVCAR8-PAX8-/- and MOSE-PAX8 when compared to control. Inhibition of Ras had a greater effect in OVCAR8-PAX8-/- while inhibition of Cdc42 had a greater effect in MOSE-PAX8. These data provide a mechanistic explanation for the role of PAX8 on increasing migration and metastasis in ovarian cancer.

Citation Format: Laura Hardy, Melissa Pergande, Angel Hernandez, Stephanie Cologna, Joanna Burdette. PAX8 increases migration and metastasis of high grade serous ovarian cancer through upregulation of Rho GTPases [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 4039.
Abstract 4043: A novel role for Hedgehog signaling in macrophage-mediated immune evasion
Authors: Hanna Ann , Chen Dongquan , Chandrashekar Darshan S. , Varambally Sooryanarayana , Samant Rajeev S. , Shevde Lalita A.
Published: August 2018

In the tumor microenvironment, breast cancer cells participate in crosstalk with the surrounding stroma. This tumor-stromal interaction forms a balance that dictates tumor suppressing or promoting response mechanisms. Macrophages in the tumor microenvironment are plastic and can mediate several functions depending on their activation states. Tumor-associated macrophages co-exist as two major phenotypes: anti-tumorigenic and immune-eliciting classically activated M1 as well as tumor-promoting and immune-suppressive alternatively activated M2 macrophages. Alternatively activated macrophages are associated with more aggressive stages and poor clinical outcomes in breast cancer patients as they suppress the tumoricidal properties of the immune system, thus facilitating tumor cell proliferation and dissemination. Hedgehog (Hh) signaling is a critical developmental pathway; its activation modulates vital cellular processes such as cell proliferation, stem-cell differentiation, and angiogenesis. While the Hh signaling pathway is normally tightly controlled, it can often be dysregulated which fosters tumorigenesis and tumor progression. Aberrant Hh signaling activation is particularly implicated in breast cancer progression and metastasis. In this study, we investigate the role of Hh signaling in polarizing breast cancer-associated macrophages toward the detrimental M2 subtype. We report that targeting different steps of the Hh signaling cascade attenuates the cytokine profile associated with M2 macrophages. Additionally, we demonstrate that Hh signaling enables the molecular mechanism responsible for alternative macrophage polarization. We furthermore identify significant shifts in immune cell populations infiltrating the primary tumor when we administer an FDA-approved Hh inhibitor to an in vivo mammary tumor model. This altered immune profile is dynamically characterized by a reduction in immune-suppressive cells concomitant with enhanced antigen presentation properties and cytotoxic immune cell infiltration, overall culminating in reduced metastasis. Thus, we describe a novel role for Hh signaling in enabling pro-tumorigenic immunity by alternatively polarizing breast cancer-associated macrophages. This study identifies a novel strategy, which has potential clinical implications to treat breast cancer through targeting aberrant tumor cell Hh activation and eliciting a robust anti-tumorigenic immune response.

Citation Format: Ann Hanna, Dongquan Chen, Darshan S. Chandrashekar, Sooryanarayana Varambally, Rajeev S. Samant, Lalita A. Shevde. A novel role for Hedgehog signaling in macrophage-mediated immune evasion [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 4043.
Abstract 4058: Race-associated breast cancer disparities: The role of CRYBB2 and its pseudogene
Authors: Barrow Maya A. , Martin Megan E. , Reaves Denise K. , Jeffcoat Breanna , Smith Ginger R. , Tarpley Michael , Williams Kevin P. , Fleming Jodie M.
Published: August 2018

Research demonstrates young African American (AA) women have higher mortality rates for triple-negative breast cancer (TNBC), despite lower incidence rates, when compared to Caucasian American (CA) women. These disparate poor patient outcomes may be attributed to access to quality care, socioeconomic status, education, or geographic location. However, numerous other studies have demonstrated that gene expression differences may also contribute to the higher mortality rates experienced by AA women, suggestive of biological differences among these two patient populations. Consequently, CRYBB2 is one gene that has consistently been associated with AA race in normal and malignant tissues. Although, its basic function in these tissues is currently unknown, higher expression of CRYBB2 is significantly correlated with poor outcome in breast, colorectal and prostate cancers in AAs. We analyzed all publically available gene expression datasets from studies focused on breast cancer health disparities and discovered that all probes detected CRYBB2 as well as its pseudogene, CRYBB2P1. It is becoming increasingly clear that pseudogenes have the ability to modify the mechanisms of their functional parental genes. However, it remains unclear whether the high expression of CRYBB2, CRYBB2P1, or both, affect cell behaviors to alter disease progression. Herein, we investigated the biological consequences of CRYBB2 and CRYBB2P1 in three basal-like TNBC cell lines (SUM159, MDA-MB-231, Hs578t) using CRISPR/Cas9 and overexpression technologies. The functional consequence of CRYBB2 and CRYBB2P1 expression were evaluated using in vitro and biochemical assays, as well as in vivo tumorigenesis studies. Our findings demonstrate overexpression of CRYBB2 and CRYBB2P1 alter cell proliferation, cytokine and growth factor production, and tumorigenesis. This data highlights the potential molecular mechanisms by which CRYBB2 and CRYBB2P1 contribute to poor patient outcome.

Citation Format: Maya A. Barrow, Megan E. Martin, Denise K. Reaves, Breanna Jeffcoat, Ginger R. Smith, Michael Tarpley, Kevin P. Williams, Jodie M. Fleming. Race-associated breast cancer disparities: The role of CRYBB2 and its pseudogene [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 4058.
Abstract 4059: Vaccination causes a clear subtype shifting of tumor infiltrate Treg in mouse breast cancer homografts
Authors: AN Xiaoyu , Ouyang Davy Xuesong , Liu Jinping , Chen Li , Li Henry Qixiang
Published: August 2018

Tumor-infiltrating lymphocytes (TIL), including CD8+ TIL-Teff (effector T-cells) and TIL-Treg (regulatory T-cells with immune suppression on self-restricted Teff), are believed to play a significant, but somewhat conflicting, role in the cancer prognosis and sensitivity to immune-oncology (I/O) treatments, including immune checkpoint inhibitors (ICI). Forkhead box P3 (FoxP3+) has long been considered a key marker of Treg while recent research has indicated significant heterogeneity of Treg with different levels of FoxP3 and the associated functional variations. The heterogeneity may explain some of the conflicting observations. The influence of the Treg heterogeneity on ICI treatment has yet to be investigated. We have previously created a mouse tumor homograft, mBR6004, by orthotopic-implanting breast adenocarcinoma spontaneously developed in MMTV-PyVT transgenic mice (GEMM) to the syngeneic FVB/N mice. The homograft has the similar growth, metastasis and histopathology as seen for the original GEMM tumors, and it responds to the CTLA-4 but not to the PD1/PDL1 inhibitors. Further, we also found that a prior vaccination treatment seemed to potentially render the tumor sensitive to PD1/PD-L1 inhibitors with increased CD8+ TIL in the treated tumors. To explore the underlying mechanism of vaccination, we are performing extensive immunophenotyping of TILs in the vaccinated tumors. Interestingly, our analysis clearly displayed a drastic shift of TIL-Treg from FoxP3lo to FoxP3hi population in the tumors with the prior vaccination as compared to the tumor without vaccination, which likely corresponds to the reported naïve FoxP3lo Treg and FoxP3hi effector Treg (eTreg). This FoxP3hi -shifted Treg is also largely absent in peripheral blood, even in the vaccinated mice, consistently to those seen in patients. Our data seem to suggest that the TIL-eTreg status, not naïve Treg, could play a critical role in tumor response to the checkpoint inhibitor treatment, and that the methods, such as vaccination to change TIL-eTreg, could be an important strategy to enhance checkpoint inhibitor treatment. In addition, the elucidation of TIL-Treg in animal model could also greatly help to investigate I/O mechanisms in the experimental settings.

Citation Format: Xiaoyu AN, Davy Xuesong Ouyang, Jinping Liu, Li Chen, Henry Qixiang Li. Vaccination causes a clear subtype shifting of tumor infiltrate Treg in mouse breast cancer homografts [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 4059.
Abstract 406: A proteome-transcriptome-miRnome integrated analysis identifies similarity between UV-exposed skin and wounding skin
Authors: Nguyen Tran N. , Rajapakshe Kimal , Avdieiev Stanislav , Nicholas Courtney , Chitsazzadeh Vida , Welsh Eric , Fang Bin , Koomen John , Coarfa Cristian , Einspahr Janine , Tsai Kenneth Y.
Published: August 2018

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer, for which long term UV exposure and chronic wounding are the dominant risk factors. Despite these clinically established connections, little is understood about the early molecular response of human skin to UV exposure and its connection to acute wounding and cuSCC. Thus, our goal is to find common and specific signatures driven by UV-exposure and wounding as a means of developing new approaches for treating and preventing cuSCC. Here, we perform integrated analyses of proteomic, RNA-seq and miR-seq on 3 datasets: (1) UV-unexposed and acute UV-exposed human skin, (2) public dataset on acute wound healing and (3) our previously published dataset on normal skin and cuSCC from humans. We find that biological signatures and processes regulated by acute UV exposure and wounding has profound similarity. miR-seq data shows that miR-223, miR-132 and miR-142 are significantly upregulated in both acute events. Combined gene set enrichment analysis shows that G-protein-coupled-receptors (GPCRs) pathways are upregulated, possibly through Gαi activation. While ECM remodeling is significantly enriched in all three datasets, gene expression regulated by PPARα is suppressed. Interestingly, upregulation of matrisome components is observed among all three datasets. This suggests that these changes are important early events that regulated by both UV-exposure and wounding which eventually can promote cuSCC initiation. Thus, our findings suggest that these common signatures can be potentially validated as chemopreventive targets for cuSCC.

Citation Format: Tran N. Nguyen, Kimal Rajapakshe, Stanislav Avdieiev, Courtney Nicholas, Vida Chitsazzadeh, Eric Welsh, Bin Fang, John Koomen, Cristian Coarfa, Janine Einspahr, Kenneth Y. Tsai. A proteome-transcriptome-miRnome integrated analysis identifies similarity between UV-exposed skin and wounding skin [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 406.
Abstract 4070: The relationship between members of the canonical NF-κB pathway, components of the tumor microenvironment and cancer-specific survival in colorectal cancer patients
Authors: Quinn Jean A. , Bennett Lindsay , Patel Meera , Loi Lynette , Frixou Mikaela , Roseweir Antonia , Park James H. , Horgan Paul G. , McMillan Donald C. , Edwards Joanne
Published: August 2018

The canonical NF-κB pathway regulates the transcription of many genes which may be involved in processes such as inflammation and proliferation, suggesting a potential link between the tumor, the tumor microenvironment and cancer-specific survival in colorectal cancer patients.

Immunohistochemistry was used to assess the expression of the upstream kinase TAK1/pTAK1 and four members of the canonical NF-κB pathway in tissue microarrays. Protein expression was determined using the weighted histoscore method. BRaf status, tumor stroma percentage, local inflammation, systemic inflammation and cancer-specific survival were examined in patients with colorectal cancer.

Cytoplasmic IKKβ was significantly associated with the inflammatory cell infiltrate (p=0.0015), systemic inflammation (p=0.03) and cancer-specific survival (p=0.046). In contrast, no significant association was found with the other three members. Cytoplasmic pTAK1 was associated with the tumor microenvironment (p=0.045) and cancer-specific survival (p=0.032). On multivariate analysis only cytoplasmic IKKβ associated with survival (HR1.75, 95%CI 1.05-291, p=0.033) and this was independent of TNM stage and markers of the tumor microenvironment. When cytoplasmic IKKβ was stratified with BRaf status (wild type (n=151) or mutant (n=48)), it was no longer associated with cancer-specific survival. However, when cytoplasmic pTAK1 was stratified with BRaf status its association with cancer-specific survival was strengthened. Cytoplasmic pTAK1 was significantly associated with cancer-specific survival in patients with wild type BRaf (p=0.014). In patients with BRaf mutations there was no significant association with cancer-specific survival (p=0.105), however the numbers were low and this is currently being investigated in a larger cohort.

The results of the present study show that high expression of cytoplasmic IKKβ was associated with decreased cancer-specific survival and with markers of the tumor microenvironment in patients with colorectal cancer. Expression of cytoplasmic pTAK1 was associated with cancer-specific survival and this was enhanced in patients with tumors expressing wild type BRaf.

Citation Format: Jean A. Quinn, Lindsay Bennett, Meera Patel, Lynette Loi, Mikaela Frixou, Antonia Roseweir, James H. Park, Paul G. Horgan, Donald C. McMillan, Joanne Edwards. The relationship between members of the canonical NF-κB pathway, components of the tumor microenvironment and cancer-specific survival in colorectal cancer 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 4070.
Abstract 4072: Direct transdifferentiation of glioblastoma cells to antigen-presenting cells: A novel immunotherapeutic approach
Authors: Sebastian Mathew , Le Son B. , Deng Changwang , Thomas Nagheme , Tran David D.
Published: August 2018

Cell-based immunotherapy has shown encouraging results in solid cancers. However, major limitations of this approach include difficulty with isolation and generation of effective antigen-presenting or immune effector cells and the high cost of cell-based vaccines. In glioblastoma (GBM), the most common and lethal brain cancer in adults, inadequate migration of immune cells across the blood-brain barrier represents another major obstacle to cell-based immunotherapy. One approach to overcome these barriers is to convert GBM cells directly into immune cells of interest. Transdifferentiation, the transformation of one cell type other than stem cells directly into another cell type without an intermediate pluripotent stage, has shown early promise in regenerative medicine, but has had a limited role as a cancer therapy, especially for solid tumors. This is in large part due to the laborious and time-consuming process of identifying master fate determinants of the cell types of interest in the transdifferentiation. To enable this novel application, we have developed and experimentally validated two tandem computational platforms, GeneRep and nSCORE. Applying this novel algorithm to large quantities of published and in-house gene expression datasets, we successfully predicted cell fate determinants of tens of different cell types with several having been validated in transdifferentiation experiments by others and us, such as the conversions of astrocytes to neuronal stem cells, of astrocytes to glioma stem-like cells, of fibroblasts to macrophages, etc. Here, we describe the process of combining a unique core set of hematopoietic stem cell, myeloid, and macrophage or dendritic cell (DC) fate determinants—some have not been described as critical in these cell fates—as predicted and highly ranked by GeneRep-nSCORE to efficiently transdifferentiate GBM cells into functional macrophages or dendritic cells, respectively, while neutralizing their malignant phenotype. This novel transdifferentiation has the potential for transformative impacts in many areas, including DC vaccine immunotherapy and cancer therapy in general, and pioneers the concept that solid tumor cells can be converted into another cell type, and not just any cell type, but one that potentially can elicit a therapeutic response against itself.

Citation Format: Mathew Sebastian, Son B. Le, Changwang Deng, Nagheme Thomas, David D. Tran. Direct transdifferentiation of glioblastoma cells to antigen-presenting cells: A novel immunotherapeutic approach [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 4072.
Abstract 4075: Role of yes-associated protein 1 (YAP1) in human pancreatic ductal adenocarcinoma initiation and progression
Authors: Yin Xue , Liu Jun , Akanuma Naoki , Nipper Michael , Wang Pei
Published: August 2018

Introduction: Understanding the molecular mechanisms underlying pancreatic ductal adenocarcinoma (PDAC) initiation and progression is an urgent need to achieve more effective diagnosis and treatment to this deadly cancer. We have developed a flow cytometry-based, high resolution lineage tracing method and 3D culture system to study PDAC initiation and progression using normal human pancreatic cells. Methods: Normal human acinar cells or cells undergoing acinar-to-ductal metaplasia (ADM, AD cells) are isolated by fluorescence-activated cell sorting (FACS), and cultured in 3D-culture system. Lentivirus carrying either YAP1-mCherry are transduced into acinar cells to study whether YAP1 is sufficient to induce ADM or is required during TGFβ-induced ADM. Lentivirus carrying either shYAP1-mCherry or shYAP1-KrasG12D-mCherry are transduced into AD cells to study whether YAP1 is required for AD cell proliferation and Kras-induced prolonged proliferation. ChIP-PCR and genome-wide ChIP-seq will be used to identify the mechanism through which YAP1 promotes PDAC development and progression. Lentivirus carrying inducible shYAP1 will be transduced into cancer producing, engineered AD cells and orthotopically transplanted into SCID mice to investigate whether inhibiting YAP1 could suppress PDAC progression. Results: We already found that TGFβ could induce ADM and oncogenic Kras could maintain the proliferation capacity of AD cells. Also, TGFβ induces YAP1 expression and TGFβ inhibitor blocks YAP1 induction during ADM process, and deleting YAP1 will suppress Kras-induced over-proliferation in AD cells, suggesting that YAP1 plays an important role in ADM, and is downstream of TGFβ as well as Kras signaling. Conclusion and significance: Successful completion of the project will give us a better understanding of how YAP1 functions in human PDAC initiation and progression. It will provide us opportunities to develop new methods for early detection and treatment of PDAC.

Citation Format: Xue Yin, Jun Liu, Naoki Akanuma, Michael Nipper, Pei Wang. Role of yes-associated protein 1 (YAP1) in human pancreatic ductal adenocarcinoma initiation and 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 4075.
Abstract 408: Evidence of DLL4, NNAT, and SLC35C2 in suppression of breast cancer initiation, growth, and metastasis
Authors: Plasterer Cody , Lemke Angela , Murphy Dana , Bergom Carmen , Joshi Amit , Rui Hallgeir , Flister Michael J.
Published: August 2018

Breast cancer affects 1 in 8 women, resulting in 40,000 deaths annually. In most cases, a single cause of breast cancer cannot be found, but rather multiple environmental and genetic factors contribute to overall disease susceptibility. This, combined with complex gene interaction in both malignant tumor cells and nonmalignant tumor microenvironment (TME) cells, poses significant challenges in sifting through the many variants that contribute to the ~31% of breast cancer risk that is heritable. Here, we narrowed the regions associated with breast cancer risk on rat chromosome 3 (RNO3) by introgressing portions of RNO3 derived from the BN rat (protective strain) onto the genomic background of the SS rat (susceptible strain). These SS.BN3 congenics were then phenotyped for DMBA-induced mammary tumor incidence, latency, and multiplicity, which revealed two loci in close proximity that contribute to mammary tumor risk: chr3:95-130Mb (QTL1) and chr3: 154-177Mb (QTL2). By comparing these data with a previous study (Flister et al. Breast Cancer Res Treat. 2017 Aug;165(1):53-64.), we concluded that QTL1 is dependent on the host TME, whereas QTL2 directly modifies breast tumorigenesis and cancer cell proliferation. By combining the congenic mapping studies with genomic and transcriptomic sequencing, and functional analysis, we have now localized the top three candidate modifiers on RNO3: DLL4 (QTL1; TME modifier), NNAT (QTL2; cancer cell modifier), and SLC35C2 (QTL2; cancer cell modifier). Collectively, these data demonstrate the effects of several novel breast cancer modifiers, as well as highlight the potential interactions between modifiers of the malignant cancer cells and the nonmalignant host TME.

Citation Format: Cody Plasterer, Angela Lemke, Dana Murphy, Carmen Bergom, Amit Joshi, Hallgeir Rui, Michael J. Flister. Evidence of DLL4, NNAT, and SLC35C2 in suppression of breast cancer initiation, growth, and metastasis [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 408.
Abstract 4089: Establishment and long-term in vitro culture of organoids derived from human biliary tract carcinomas
Authors: Saito Yoshimasa , Muramatsu Toshihide , Sugiyama Yuko , Uchida Ryoei , Furukawa Ryo , Yoshikawa Nao , Yamaguchi Tomoko , Kanai Yae , Saito Hidetsugu
Published: August 2018

Background Biliary tract carcinomas (BTCs) are epithelial malignancies arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. Although patients with inoperable BTCs generally receive a chemotherapy regimen of gemcitabine and cisplatin, the effect of these drugs is limited and the 5-year survival rates of patients are very low. The newly developed 3D culture system known as “organoid culture” allows long-term expansion of stem cells into budding cyst-like structures (organoids) with properties resembling those of the original tissues. The aim of this study is to establish in vitro preclinical models for patients with BTCs using the organoid culture technology.

Methods We established organoids using cancer tissues obtained from patients with BTCs. Alterations of genome, gene expression profiles including microRNAs and drug sensitivity were analyzed in BTC organoids.

Results We were successful in establishment and long-term in vitro culture of five organoid lines derived from human BTCs including intrahepatic cholangiocarcinoma, gallbladder cancer and neuroendocrine carcinoma of the ampulla of Vater. These BTC organoids can be stably cultured over a year, whereas organoids derived from non-cancer bile duct and gallbladder tissues ceased proliferating after 15 passages or six months. H&E staining and immunohistochemistry of BTC organoids and the primary tissues demonstrated that BTC organoids closely recapitulate the histopathological features of the primary tumors. The tumor suppressor microRNA miR-34a was suppressed in organoids derived from one case of intrahepatic cholangiocarcinoma, and enforced expression of miR-34a markedly suppressed the growth of these organoids with down-regulation of its target oncogenes. We also examined the effect of the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) on BTC organoids. Treatment with 5-Aza-CdR suppressed the proliferation of all BTC organoids by inducing an anti-viral immune response including activation of interferon-responsive genes.

Conclusions Studies using organoids derived from various BTC cases could potentially pave the way for development of precision medicine for patients with BTCs. These preclinical models for refractory cancers may fill the gap between basic cancer research and patients trials and allow personalized cancer therapy.

Citation Format: Yoshimasa Saito, Toshihide Muramatsu, Yuko Sugiyama, Ryoei Uchida, Ryo Furukawa, Nao Yoshikawa, Tomoko Yamaguchi, Yae Kanai, Hidetsugu Saito. Establishment and long-term in vitro culture of organoids derived from human biliary tract carcinomas [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 4089.
Abstract 4095: Active Kras induced metaplasia progression toward gastric neoplasia is suppressed by MEK inhibition during gastric carcinogenesis
Authors: Choi Eunyoung , Engevik Amy C. , Goldenring James R.
Published: August 2018

Intestinal type gastric cancer develops within metaplastic mucosal fields and development of gastric neoplasia represents the most prominent example of gastric cancer developing within the morphologic changes of metaplasia. Recent investigations have noted activation of Ras activity in up to 40% of human gastric cancers and suggest. Our recent study has suggested that chief cells might be an origin of gastric cancer using a mouse model, Mist1-CreERT2;LSL-KRas(G12D) mouse (Mist1-Kras).  The Mist1-Kras mice rapidly developed SPEM and IM within 3 months and invasive metaplasia within 4 months following induction of constitutively-active Kras(G12D) expression in the chief cells in the mouse stomach. We also observed regression of metaplasia by targeting downstream mediators of Kras signaling using a MEK inhibitor, Selumetinib. We therefore have now investigated how the progression and proliferation of metaplasia can be controlled. In this study, we have established metaplastic organoid lines (Meta3 and Meta4) from Mist1-Kras mouse stomach mucosa at 3 or 4 months after tamoxifen induction. We examined these organoids to understand the mechanisms of metaplasia progression through MEK inhibition.  Both Meta3 and Meta4 organoids have formed distinguishable glandular structures in 3D cultures and maintained a stable phenotype observed in metaplastic glands in the Mist1-Kras mouse stomachs. The MEK inhibition with Selumetinib in the metaplastic organoids inhibited growth, but promoted differentiation of metaplastic cells toward intestinal absorptive cells with elaboration of an apical brush border. Single cell RNA sequencing data revealed that Meta-4 have distinct characteristics that differ from Meta3. In 3D culture, Meta4 organoids developed more aggressive phenotypes than Meta3 and expressed Cortactin, which has critical roles in cancer cell migration and metastasis. Cortactin localized at the lamellipodia in monolayer culture of Meta4. Meta4 organoids treated with Selumetinib produced significantly fewer colonies in soft agar and resulted in loss of lamellipodia localization of Cortactin. Our results indicate that gastric carcinogenesis can be controlled by a MEK inhibitor, Selumetinib, and support the concept that growth of pre-neoplastic metaplasia and early stage gastric cancer in humans might be controlled with administration of Selumetinib.

Citation Format: Eunyoung Choi, Amy C. Engevik, James R. Goldenring. Active Kras induced metaplasia progression toward gastric neoplasia is suppressed by MEK inhibition during gastric carcinogenesis [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 4095.
Abstract 4098: New strategy of drug response assessment using PDX organoid platform for non-resectable pancreatic cancer
Authors: Jeon A-Ra , Choi Sun Il , Park Sang-Jae , Han Sung-Sik , Kong Sun-Young , Kim Min Kyeong , Lee Yu-sun , Im Jieun , Lee Min Kyeong , Park Sang Hyun , Kim Joon-Ki , Yoon Kyong-Ah , Koh Young-Hwan , Lee Ju Hee , Lee Woo Jin , Woo Sang Myung , Kim Yun-Hee
Published: August 2018

To develop an efficient drug screening platform which overcomes the difference of drug response between initial screening and clinical trial stage is a pivotal issue for drug discovery. The patient-derived Xenograft (PDX) model has been reported as a screening system to reflect the microenvironment and heterogeneity of tumor. However, in pancreatic cancer that 80 % of patients is non-resectable, PDX is not be suitable for an initial screening model in terms of economic- and time cost of mouse-based amplification system as well as the lack of obtaining pancreatic tumor tissue from fine needle biopsy or percutaneous gun. To overcome this limitation, here we newly suggested organoids system, miniature organ culture on a dish, that are generated from tumor tissues of orthotopic PDX model, which has the advantages of reflection of each patient's characteristics as well as amplification of limited tumor tissue. Besides, it is possible to screen of drug responsibility with a little number of cells. 12 organoids derived from PDX using needle or gun biopsy tumor tissues showed EpCAM overexpression and each unique morphological phenotype. Moreover, from drug responsibility test, H #43 and H #44, an organoids derived from a gemcitabine-sensitive patients, were highly responsible to gemcitabine, whereas the organoids from gemcitabine-resistant patients, G #20 and H #19 showed a strong resistance to gemcitabine as measuring the IC50 value. In addition, combined treatment with gemcitabine and abraxane to the G #13 model which has no clinical information of drug response due to early death, it inhibited organoid formation significantly, showing a combination index below 1, which was proved through in vivo (PDX) validation. Taken together, the PDX-Organoid system might be able to reflect primary tumor characteristics as well as to overcome the quantitative limitations of the specimen and time cost, and thereby it is possible to predict drug response early in vitro, making it very efficient as an anti-cancer drug development platform for pancreatic cancer.

Citation Format: A-Ra Jeon, Sun Il Choi, Sang-Jae Park, Sung-Sik Han, Sun-Young Kong, Min Kyeong Kim, Yu-sun Lee, Jieun Im, Min Kyeong Lee, Sang Hyun Park, Joon-Ki Kim, Kyong-Ah Yoon, Young-Hwan Koh, Ju Hee Lee, Woo Jin Lee, Sang Myung Woo, Yun-Hee Kim. New strategy of drug response assessment using PDX organoid platform for non-resectable pancreatic 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 4098.