bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3670: Development of cfDNA-based methylation assay for early cancer detections
Authors: Xiang Binggang , Zhao Zhixin , Stevens Maya , Dai Chao , Xie Feng , Wang Xiaohong , Zhou Kemin , Jia Shidong , Du Pan , Yu Jianjun
Published: September 2020

Introduction: DNA methylation is the essential epigenetic mechanism of cell regulation, and one of the earliest signatures in cancer development. Blood-based DNA methylation has been tested for cancer early detection. Several methods have been used for DNA methylation sequencing, which include whole genome bisulfite sequencing (WGBS) and antibody-dependent DNA immunoprecipitation (MeDIP). However, WGBS has known issues of DNA loss and fragmentations, and MeDIP only captures the methylated DNA regions and are biased. Therefore, methods that can accurately quantify DNA methylation status while keeping DNA less damaged and loss are highly needed, especially for liquid biopsy at early cancer stages (due to its low cfDNA amount).

Methods: We developed a probe capture-based cfDNA methylation assay using proprietary in-house developed liquid biopsy NGS platform and DeepSEA software algorithm. Analytic assay validation has been conducted using reference materials with known methylation profiles. Both cell line gDNA with known methylation profiles and plasma cfDNA from patients were used for this methylation study. Methylation profiles, DNA damages, and losses were evaluated and compared. Fragment distribution and copy number variations (CNV) were compared between untreated and treated samples.

Results: In this study, we developed a cfDNA-based methylation assay that measures DNA methylation status and genomic profiling simultaneously. Using both cell line gDNA and plasma cfDNA, the assay detects both methylation change and genomic alterations, including fragment size distribution and CNV in a single test.

Discussion: The successful development of cfDNA methylation assay could help identify cancer at its early stage. It also enables the detection and monitoring of minimal residual disease (MRD) and in obtaining information on therapeutic targets and resistance mechanisms relevant to the management of personalized cancer care.

Citation Format: Binggang Xiang, Zhixin Zhao, Maya Stevens, Chao Dai, Feng Xie, Xiaohong Wang, Kemin Zhou, Shidong Jia, Pan Du, Jianjun Yu. Development of cfDNA-based methylation assay for early cancer detections [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3670.
Abstract 3685: Sustained Ret expression during mammary gland post-lactation induces premature involution and enhances cancer potential
Authors: Vallone Sabrina A. , Solá Martín García , Cardiff Robert D. , Meiss Roberto P. , Chodosh Lewis A. , Shere-Levy Carolina , Kordon Edith C C. , Hynes Nancy E. , Gattelli Albana
Published: September 2020

Loss of normal development is a hallmark of cancer. Thus, understanding the mechanisms of tissue-specific developmental regulation and the changes that occur during tumorigenesis may provide insights of both diagnostic and therapeutic importance. In breast cancer, several members of the receptor tyrosine kinases (RTK) family that are well known to promote aggressive breast cancers also have roles in normal breast. We found that Ret, a RTK member, is normally expressed in the mouse glands in lactation. We determined that inhibition of Ret activity in vivo does not alter lactation, however impacts in the transition to involution. Involution is the period with high inflammation which returns the lactating mammary gland to a quiescent state after weaning. Involution has been well described as a post-lactation stage that drives cancer progression. Ret is overexpressed in about 40% of human breast tumors. Previously, using a doxycycline-inducible transgenic mouse model (Ret/MTB) we determined that chronic expression of Ret is oncogenic in the mammary epithelium. However, the stage of development at which Ret expression results in increase mammary tumor incidence has not been identified. To address this, we used the Ret/MTB system, to conditionally overexpress Ret during discrete periods of mammary gland development. We found that Ret is required for efficient transition to involution. We determined that the induction of Ret in Ret/MTB females promotes the expression of factors that drives involution, including premature Stat3 activation. RNA-seq data in Ret-overexpressing glands is supporting these findings, which were confirmed by several techniques. In addition, sustained expression of Ret during post-lactation enhances cancer potential showing a significant increase in pre-neoplastic lesions, defective milk recycling and disrupting Stat3 signaling. These results demonstrate that Ret deregulation increases cancer potential in post-lactation and might be considered as a prognostic marker for post-partum breast cancer.

Citation Format: Sabrina A. Vallone, Martín García Solá, Robert D. Cardiff, Roberto P. Meiss, Lewis A. Chodosh, Carolina Shere-Levy, Edith C C. Kordon, Nancy E. Hynes, Albana Gattelli. Sustained Ret expression during mammary gland post-lactation induces premature involution and enhances cancer potential [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3685.
Abstract 3691A: Targeting MARK2-HDAC-YAP signaling to overcome paclitaxel resistance in PDAC
Authors: Zeng Yongji , Chen Yuanhong , Dong Jixin
Published: September 2020

Nanoparticle albumin-bound (Nab)-paclitaxel (taxol) combined with gemcitabine has been approved as a first-line chemotherapy for advanced pancreatic ductal adenocarcinoma (PDAC) patients. However, the response rate is still very low due to the occurrence of drug resistance and attempts in reversing the resistance have not been successful in the clinic.

MARK (microtubule affinity-regulating kinase) proteins (MARK1-4) are critical for microtubule dynamics by modulating MAPs (microtubule-associated proteins). They are members of the AMPK/Snf1 family and control cell polarity and asymmetric cell division. Dysregulation of MARKs has also been associated with pathological conditions such as cancer and Alzheimer disease. Several recent studies showed that MARK family members are important regulators of Hippo-YAP (Yes-associated protein) signaling, which is critical in cancer development, drug resistance, and stem cell biology.

Through Phos-tag-based kinome-wide screens, we identified MARK2 as a critical determinant for mitotic progression and paclitaxel chemosensitivity. MARK2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) during paclitaxel-induced mitotic arrest and normal mitosis. MARK2 inhibition did not affect growth under normal conditions, but greatly sensitized PDAC cells to paclitaxel-induced apoptosis. Mechanistically, our findings also suggest that MARK2 controls paclitaxel chemosensitivity by regulating class IIa HDACs (histone deacetylase). MARK2 directly phosphorylates HDAC4 on multiple sites upon paclitaxel treatment. Inhibition (with knockdown or inhibitors) of HDACs significantly enhanced paclitaxel-induced cell death in a MARK2-phosphorylation dependent manner in PDAC cells. MARK2-phosphorylated HDAC4 positively regulates YAP (the transcriptional coactivator of the Hippo pathway) activity and controls expression of YAP target genes induced by paclitaxel. MARK2, class IIa HDACs, and YAP expression levels are upregulated and positively correlated in PDAC patients. Importantly, combination of HDAC inhibition (Vorinostat/SAHA, FDA-approved) and paclitaxel overcomes paclitaxel-resistance in preclinical PDAC animal models. Together, our findings reveal the MARK2-HDAC-YAP axis as a druggable target for overcoming paclitaxel resistance in PDAC.

Citation Format: Yongji Zeng, Yuanhong Chen, Jixin Dong. Targeting MARK2-HDAC-YAP signaling to overcome paclitaxel resistance in PDAC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3691A.
Abstract 3721: Relationship between insulin resistance and chemosensitivity of high grade bone sarcoma
Authors: Boudou-Rouquette Pascaline , Gataa Ithar , De Percin Sixtine , Tlemsani Camille , Biau David , Dumaine Valerie , Audard Virginie , Larousserie Frédérique , Anract Philippe , Jouinot Anne , Goldwasser François
Published: September 2020

Background: Neoadjuvant chemotherapy with intercalated surgery is the standard of care for resectable high-grade bone sarcoma (BS) but identification of biomarkers for chemosensitivity prediction is still needed. Insulin-like Growth Factor (IGF) pathway was first linked with sarcoma biology approximately 30 years ago (El-Brady OM, 1990).

Methods: We recruited 138 pts with newly diagnosed primary localized or metastatic BS. We measured fasting blood glucose and insulin, body mass index (BMI). The HOMA-IR (Homeostasis Model Assessment) score ((Glucose (mmol/l) x Insulin) / 22.5), which has been proven to reliably detect insulin resistance IR was calculated, and patients with HOMA-IR > 2.6 were considered as IR. We collected characteristics usually associated with insulin resistance: age, obesity, diabetes, dyslipidemia and hypertension.

Results: Out of 138 pts, 50 (36%) were insulin resistant: HOMA-IR >2.6. Thirty-two IR patients (64%) had no cardiovascular risk factor. In univariate analysis, IR was associated with obesity (BMI > 30 kg/m2)(22% vs 3%, p=0.0015), but not with age (median 36,8 vs 41 years, p=0.19). IR was more prevalent in Ewing sarcoma (14/29; 48%) and chondrosarcoma (7/16; 44%) followed by osteosarcoma (25/67; 37%) and other rare bone sarcomas (4/26; 15.3%), but not significantly (p=0.097). After neoadjuvant chemotherapy and surgery of Ewing tumor and osteosarcoma, pathologic tumor necrosis was higher in IR pts (88.9 vs 75%, p= 0.028).

Conclusion: Our results suggest that metabolic profile may help to discriminate best candidates for neoadjuvant chemotherapy in high-grade bone sarcomas. Further studies are needed to confirm and explain these results.

Citation Format: Pascaline Boudou-Rouquette, Ithar Gataa, Sixtine De Percin, Camille Tlemsani, David Biau, Valerie Dumaine, Virginie Audard, Frédérique Larousserie, Philippe Anract, Anne Jouinot, François Goldwasser. Relationship between insulin resistance and chemosensitivity of high grade bone sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3721.
Abstract 3732: Characterization of tumor relevant isocitrate dehydrogenase 1 (IDH1)
Authors: Coleman Nalani J. , Thornberg Ella , Caliger Danielle , Luna Lucas , Sohl Christal D.
Published: September 2020

Isocitrate dehydrogenase 1 (IDH1) is found commonly mutated in grade II and III gliomas and secondary glioblastomas. Normally, IDH1 catalyzes the conversion of isocitrate and NADP+ to α-ketoglutarate and NADPH. However, in mutated form, IDH1 catalyzes α-ketoglutarate and NADPH into NADP+ and D-2-hydroxyglutarate, an oncometabolite. D-2-hydroxyglutarate is toxic to cells and drives a number of pro-tumorigenic pathways. To understand the molecular mechanisms of how mutated IDH1 can lead to brain cancer, we created point mutations in IDH1 to understand the effects on enzyme activity. Site-directed mutagenesis was used to generate mutations at residue 132, the most common site of mutations in cancer patients. Wild type (WT) IDH1 and mutant IDH1 were heterologously expressed in bacteria . Following purification using affinity column chromatography, steady-state kinetic assays were used to compare the catalytic activity of WT versus mutant IDH1. By elucidating the types of mutations at R132 that facilitate D2HG production, we can have a better understanding of how IDH1 mutations affect prognosis and therapeutic response.

This work was funded by a Research Scholar Grant, RSG-19-075-01-TBE, from the American Cancer Society (CDS), National Institutes of Health R00 CA187594 (CDS), National Institutes of Health U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), and the California Metabolic Research Foundation (SDSU).

Citation Format: Nalani J. Coleman, Ella Thornberg, Danielle Caliger, Lucas Luna, Christal D. Sohl. Characterization of tumor relevant isocitrate dehydrogenase 1 (IDH1) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3732.
Abstract 3748: Tyrosine kinase inhibition of fibroblast growth factor receptor 1 in advanced prostate cancer
Authors: Bengtsson Louise Medina , Labanca Estefania , Navone Nora
Published: September 2020

Advanced prostate cancer (PCa) is one of the leading causes of cancer death in men in the United States. The FGF/FGFR1 signaling axis has been implicated in the progression of PCa. Different isoforms of FGFR1 have been described in PCa (e.g., alpha and beta), which might account for the typical heterogeneity of PCa. The isoforms may also mediate different clinical responses to treatments targeting this pathway. Pan-FGFR tyrosine kinase inhibitor JNJ-42756493 (Janssen) has been demonstrated to have antiproliferative effects in FGFR pathway-activated cancer cell lines and in vivo mouse antitumor activity. Our goal was to study the effects of JNJ-42756493 in PCa growth and signaling in vitro and establish whether these effects differ according to the FGFR1 isoform (alpha or beta) expressed. Using transfected bone-derived PCa cell lines and patient-derived xenografts (PDXs), we measured the effects of FGFR1 expression on cell proliferation and activation of the FGFR1-pMAPK signaling pathway. We then performed drug treatment experiments on FGFR1-expressing sublines and high-FGFR1 PDXs and observed the response in FGFR1-pMAPK signaling pathway activation. Our results showed that FGFR1 isoforms alpha and beta trigger different biological effects (i.e., proliferation), possibly contributing to the heterogeneity of PCa. We found that targeting the tyrosine kinase domain of FGFR1 with JNJ-42756493 results in pMAPK inhibition in PCa sublines. These findings indicate that the presence of specific FGFR1 variants should be considered for treatment selection in advanced PCa. The results also suggest that FGFR1-pMAPK pathway blockade by JNJ-42756493 should be further studied as a strategy to guide the development of therapies to hinder PCa progression.

Citation Format: Louise Medina Bengtsson, Estefania Labanca, Nora Navone. Tyrosine kinase inhibition of fibroblast growth factor receptor 1 in advanced prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3748.
Abstract 3750: STEAP2 is upregulated and necessary for hepatocellular carcinoma progression via increased copper levels and stress-activated MAP kinase activity
Authors: Zeballos Carla R. , Easley Acarizia , Bouamar Hakim , Zheng Guixi , Gu Xiang , Junhua Yang , Chen Yidong , Cigarroa Francisco , Sun LuZhe
Published: September 2020

To understand potential molecular mechanisms that drive hepatocellular carcinoma (HCC) progression, we performed RNA sequencing from paired adjacent non-tumor liver and HCC tumor tissues of nine local HCC patients which revealed significant involvement of a gene coding for Six Transmembrane Epithelial Antigen of Prostate 2 (STEAP2) protein. STEAP2 is a metalloreductase involved in the reduction and indirectly, uptake, of iron and copper ions. We found significantly higher levels of total copper in HCC tissues than those in paired adjacent non-tumor tissues. The upregulation of STEAP2 expression in HCC was confirmed at its protein level and in TCGA and other published HCC gene expression datasets. Hepatic copper overload is associated with Wilson's disease and a known risk factor for HCC, therefore, we hypothesize that STEAP2 upregulation and copper accumulation contribute to HCC progression.

Paired HCC and adjacent non-tumor tissues were collected for RNA sequencing, metal ion measurement, and measurements of STEAP2 levels with RT-qPCR and Western blot. Public HCC datasets were queried for STEAP2 expression in HCC and non-tumor tissues. HCC cell lines with knockdown (KD) and overexpression (OE) of STEAP2 were created to perform mechanistic studies including measurements of copper levels and MAP kinase activities, cell proliferation, migration, and anchorage independent growth in vitro and tumor growth in vivo.

STEAP2 is significantly upregulated in various HCC gene expression datasets; its expression is positively associated with tumor grade. STEAP2 KD in HCC cell lines decreased cell growth, migration, invasion, and xenograft tumor growth, while STEAP2 OE showed opposite effects. STEAP2 KD in HCC cells also reduced intracellular copper levels and activation of stress-activated MAP kinases including p38 and JNK. Treatment with copper rescued the reduced HCC cell migration due to STEATP2 KD and activated p38 and JNK. Furthermore, treatment with p38 or JNK inhibitors significantly inhibited cell migration. Thus, STEAP2 appears to play a malignant-promoting role in HCC cells by driving migration/invasion via increased copper levels and MAP kinase activities. Our study uncovered a novel molecular mechanism contributing to HCC malignancy and a potential therapeutic target for HCC treatment.

Citation Format: Carla R. Zeballos, Acarizia Easley, Hakim Bouamar, Guixi Zheng, Xiang Gu, Yang Junhua, Yidong Chen, Francisco Cigarroa, LuZhe Sun. STEAP2 is upregulated and necessary for hepatocellular carcinoma progression via increased copper levels and stress-activated MAP kinase activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3750.
Abstract 379: Myeloperoxidase-produced HOCl is a paracrine effector linking myeloid cells to NF-κB signaling in melanoma, mediating anti-tumor responses during early melanoma progression
Authors: Liu Tracy W. , Gammon Seth T. , Yang Ping , Fuentes David T. , Piwnica-Worms David
Published: September 2020

The myeloperoxidase (MPO) system of myeloid-derived cells (MDCs) is central to cellular innate immunity and the microbicidal activities of phagocytes, while also impacting the pathogenesis of many disorders, including cancer. Upon MDC activation, MPO is secreted into phagosomes, catalyzing the production of hypochlorous acid (HOCl). HOCl is a potent chlorinating oxidant with significant biological effects, but the mechanisms by which HOCl impacts cancer growth remain unresolved. Herein, we demonstrate that the myeloid lineage-restricted MPO-HOCl system has anti-tumor effects during early melanoma progression. Orthotopic melanomas grew slower in immuno-competent host mice wild type for MPO+/+ compared to syngeneic MPO-null (MPO-/-) animals. Using a specific and potent inhibitor of MPO, 4-aminobenzoic acid hydrazide (4-ABAH), melanoma tumor growth in MPO+/+ mice continuously administered 4-ABAH, pharmacologically mimicked the MPO-/- phenotype. Real-time intravital tumor imaging in vivo using skinfold window chamber animal models as well as live cell co-culture studies revealed a cell-cell proximity-dependent association between MDC-derived MPO activity and blockade of ligand-induced IκBα degradation in tumor cells. Real-time bioluminescent imaging and quantitative monitoring of the canonical NF-κB pathway was assessed in melanoma cells stably expressing bioluminescent reporters: a transcriptionally-activated NF-κB-promoter-driven Firefly luciferase (FLuc) reporter or a fusion reporter under the control of an NF-κB-responsive promoter fused to IκBα-FLuc reporter. HOCl was shown to directly inhibit IκB kinase (IKK) activity, thereby decreasing NF-κB transcriptional activation within melanomas, increasing circulating levels of the myeloid-attracting cytokines CXCL1 and CXCL5, enhancing local infiltration of CD8+ cytotoxic T cells, and dampening tumor growth. Overall, these data reveal a novel role for MDC-derived HOCl as a small molecule paracrine signaling factor that trans-inhibits IKK in melanoma cells, mediating anti-tumor responses during early tumor progression.

Citation Format: Tracy W. Liu, Seth T. Gammon, Ping Yang, David T. Fuentes, David Piwnica-Worms. Myeloperoxidase-produced HOCl is a paracrine effector linking myeloid cells to NF-κB signaling in melanoma, mediating anti-tumor responses during early melanoma progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 379.
Abstract 3833: Epigenetic regulation of cell state transitions and the therapeutic implications in breast cancer
Authors: Toh Li Ping , Loo Ser Yue , Tam Wai Leong
Published: September 2020

During cancer progression, cells would reversibly transit among different cell states that differ in their competence to contribute towards tumor growth, survival, and metastasis. The activation of epithelial-mesenchymal transition (EMT) program in cancer cells has often been attributed to enhanced tumorigenic potential and a gain of therapeutic resistance. We hypothesized that by promoting mesenchymal-epithelial transition (MET), the reverse process of EMT, it could induce a loss of tumorigenic potential and increase susceptibility to standard chemotherapeutics. As EMT is a dynamic and reversible process, epigenetic regulations such as histone modifications and DNA methylation are important in regulating and reprogramming the expression of EMT-responsive genes. Hence, we seek to understand how the epigenetic landscape is altered during transitions between cell states. To understand the molecular link between epigenetic regulations and cell state transitions in cancer cells, a high-throughput library screen on epigenetic modifiers was performed. Epigenetic modifying compounds that can induce MET in basal-like breast cancer cells were uncovered. In vitro and in vivo data demonstrate that compounds targeting the non-BET bromodomains were able to promote MET, induce a loss of stem-like properties, and decrease the tumorigenicity of basal-like breast cancer cells. Using various next-generation sequencing approaches, we also elucidated the mechanisms of how targeting non-BET bromodomains would regulate cell state transitions in breast cancers. While epigenetic therapies are effective in the treatment of hematological malignancies, its application in solid tumors is still limited. Here, we propose the possible use of an epigenetic MET inducer for differentiation therapy of the aggressive basal-like breast cancers.

Citation Format: Li Ping Toh, Ser Yue Loo, Wai Leong Tam. Epigenetic regulation of cell state transitions and the therapeutic implications in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3833.
Abstract 3838: HDAC5-mediated deacetylation promotes SATB1 oncogenic functions
Authors: Sharma Shalakha , Das Dr. Sanjeev
Published: September 2020

HDAC5, a class IIa histone deacetylase is a prominent transcriptional regulator. The functions of HDAC5 have been shown to be regulated via phosphorylation-dependent nuclear-cytoplasmic shuttling. Phosphorylation of two conserved Ser259 and Ser498 residues by calcium/calmodulin dependent protein kinase promotes HDAC5 nuclear export. Dysregulation of HDAC5 has been implicated in development of many pathogenic conditions including cardiac hypertrophy and cancer. However, the mechanisms underlying the role of HDAC5 in tumorigenesis are not well understood. In order to explore the role of HDAC5 in tumorigenesis, we performed a proteomic screen and identified several interacting proteins of HDAC5. In vitro and in vivo studies further confirmed SATB1 as a novel interacting partner of HDAC5. SATB1 (Special AT rich binding protein 1) is a global genome organizer that recruits chromatin remodeling proteins to epigenetically regulate several genes. It has been recently reported to be overexpressed in various cancers and associated with malignant behavior of cancer cells. Our studies revealed SATB1 as a novel substrate of HDAC5-mediated deacetylation during early phase of genotoxic stress. We also mapped the site of deacetylation to the highly conserved K411 residue of SATB1. Upon prolonged genotoxic stress when HDAC5 undergoes nuclear export, SATB1 acetylation was observed. Thus, we screened a shRNA library targeting nuclear histone acetyltransferases and identified Tip60 as the cognate histone acetyltransferase involved in SATB1 acetylation at K411 residue. Next we performed qPCR array and found SATB1 upregulates the expression of genes involved in tumor suppression in acetylation-dependent manner. Increased SATB1 acetylation leads to decreased cell proliferation capacity, migration and invasiveness of tumor cells. Furthermore, studies in mouse tumor models confirmed that HDAC5 promotes SATB1-mediated tumorigenesis. Taken together, our findings add to the substrate repertoire of HDAC5 and provides key insights into SATB1-mediated tumorigenesis.

Citation Format: Shalakha Sharma, Dr. Sanjeev Das. HDAC5-mediated deacetylation promotes SATB1 oncogenic functions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3838.
Abstract 3840: KLF5 acetylation promotes bone metastatic growth of prostate cancer by activating CXCR4/IL-11 and subsequent osteoclast differentiation
Authors: Zhang Baotong , Li Yixiang , Wu Qiao , Xie Lin , Barwick Benjamin , Dong Jin-Tang
Published: September 2020

Advanced prostate cancer most commonly spreads to bones. Although denosumab or zoledronic acid is recommended for prostate cancer patients with bone metastases to prevent or delay skeletal-related events, no significant benefits in terms of cancer-specific and overall survival have been observed. Some chemotherapies are available for the treatment of bone metastasis, but eventually all such tumors develop resistance. Additionally, the underlying mechanisms of prostate cancer bone metastasis and its resistance to chemotherapy are still poorly understood. In this study, we report that TGF-β-induced acetylation of the basic transcription factor Krüppel-like factor 5 (KLF5) frequently occurred in prostate cancer cells located in both the mouse and human bone microenvironments. Using multiple in vitro and in vivo systems, we demonstrated that tumor cells expressing acetylated KLF5 (Ac-KLF5) enhanced prostate cancer-induced bone lesions by promoting osteoclast differentiation. As revealed by RNA-Seq and ChIP-Seq analyses, Ac-KLF5 transcriptionally activated CXCR4 expression, the silence of which attenuated Ac-KLF5-enhanced osteoclast differentiation. A further screening on the paracrine signaling indicates that IL-11 mediated the promoting effect of the Ac-KLF5/CXCR4 axis on osteoclast differentiation. These findings indicate that the TGF-β/Ac-KLF5/CXCR4/IL-11 signaling axis from bone to tumor and then to bone again is a crucial pathway responsible for TGF-β mediated vicious cycle in bone metastasis. More importantly, targeting CXCR4 using FDA approved drugs AMD3100 selectively sensitizes Ac-KLF5-expressing tumors and bone metastases to docetaxel treatment. These findings provide a rationale for targeting Ac-KLF5/CXCR4 signaling as a novel therapeutic strategy in advanced prostate cancer patients with bone metastases.

Citation Format: Baotong Zhang, Yixiang Li, Qiao Wu, Lin Xie, Benjamin Barwick, Jin-Tang Dong. KLF5 acetylation promotes bone metastatic growth of prostate cancer by activating CXCR4/IL-11 and subsequent osteoclast differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3840.
Abstract 388: Antigen-loaded Fe3O4 nanoparticles for the stimulation and tracking of dendritic cell based vaccine
Authors: Qi Yafei , Du Yang , Tian Jie , Jin Zhengyu , Li Yuan
Published: September 2020

Background and purpose:Dendritic cells (DCs), as the antigen-presenting cells, play important roles in immunotherapy. The antigen uptake, cross-presentation, maturation and lymph node migration of DCs are crucial steps for immune system activation. Herein, we aim to construct a novel Fe3O4@SIINFEKL-TAT nanoparticle, which mainly consists of Fe3O4 nanosphere core and transmembrane peptide TAT on the shell, and also with the model antigen SIINFEKL peptide, a peptide of the ovalbumin (OVA) antigen. To explore the feasibility of Fe3O4@OVA-TAT to stimulate the maturation of DCs and the in vivo tracking of DCs using MRI-MPI dual-modal imaging.

Methods: On the one hand, TAT peptide facilitates the uptake of the Fe3O4@OVA-TAT nanoparticles into DCs, and subsequently the OVA antigens are cross-presented to stimulate DCs maturation. On the other hand, Fe3O4 nanosphere core can function for both magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) dual-modality imaging, which can realize the in vivo dynamic tracking of DCs migration to lymph nodes.

Results: The laser confocal microscope experiments confirmed that Fe3O4@OVA-TAT can be taken up through the early endosome-late endosome pathway, but the fluorescent signal of the imaging probe is located outside the lysosome after 4 h, which suggested that Fe3O4@OVA-TAT could escape lysosomal uptake and cross-present. The CD80 and CD86 flow cytometry results showed that Fe3O4@OVA-TAT, LPS, and OVA can stimulate 90%, 60%, and 40% of BMDCs to mature, respectively (p < 0.05). After the Fe3O4@SIINFEKL-TAT labeled DCs was injected into the mouse footpad, the MRI T2WI signal decreased and the MPI signal increased in the popliteal and inguinal lymph nodes 24 h post-injection, suggesting that our DC vaccine can efficiently migrate to the lymph node.

Conclusions: Our study showed that Fe3O4@SIINFEKL-TAT nanoparticles can stimulate and trace DCs in in vivo, which pave the way for further development and precision application of DC vaccine in both preclinical and clinical settings.

Citation Format: Yafei Qi, Yang Du, Jie Tian, Zhengyu Jin, Yuan Li. Antigen-loaded Fe3O4 nanoparticles for the stimulation and tracking of dendritic cell based vaccine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 388.
Abstract 3905: Human liver organoids for disease modeling of fibrolamellar hepatocellular carcinoma
Authors: Croteau Nicole J. , Requena David , Lalazar Gadi , Ng Denise , Levin Solomon , Shebl Bassem , Wang Ruisi , Hammond William J. , Saltsman James A. , Gehart Helmuth , Clevers Hans , LaQuaglia Michael P. , Simon Sanford
Published: September 2020

Fibrolamellar Hepatocellular Carcinoma (FLC) is a usually lethal liver cancer affecting adolescents and young adults without previous liver disease. FLC is characterized by a ~400kb nucleotide deletion resulting in a fusion of a heatshock protein cofactor, DNAJB1, with the catalytic subunit of protein kinase A (PRKACA). One limitation to the study of this disease is the lack of in vitro models. Organoids have shown utility as a model system for studying many diseases, including cancer. This study describes the development and characterization of FLC tumor organoids. With IRB approval, normal liver and FLC tumor tissue was obtained from patients undergoing surgical resection. Liver cells were isolated from the tissue samples and placed into a 3D basement membrane matrix and the media supplemented with growth factors specific to promote liver organoid expansion. Normal and tumor organoids were analyzed by microscopy for morphology, by PCR for presence of the DNAJB1-PRKACA chimeric transcript, by Western blot for chimeric protein expression, and by RNA sequencing for transcriptomic changes. Eight organoid lines have been developed from normal liver and eight from FLC tumor tissue, including multiple independent organoids developed from different metastases from the same patient. A distinct morphological difference between normal liver organoids and FLC tumor organoids can be seen with either brightfield microscopy or with H&E. The DNAJB1-PRKACA chimeric transcript was detected in all of the tumor tissue and tumor organoids by RT-PCR and not detected in any of the normal tissue or organoids. The presence of the DNAJB1-PRKACA fusion protein was verified by Western blot in tumor organoids and tissue but not detectable in the normal tissue. The RNA sequencing analysis shows clustering of tumor organoids with tumor tissue. Tumor organoids injected subcutaneously in mice grew as tumors. Now that the normal and tumor organoids have been developed and validated, they are being used for screening for potential therapeutics.

Citation Format: Nicole J. Croteau, David Requena, Gadi Lalazar, Denise Ng, Solomon Levin, Bassem Shebl, Ruisi Wang, William J. Hammond, James A. Saltsman, Helmuth Gehart, Hans Clevers, Michael P. LaQuaglia, Sanford Simon. Human liver organoids for disease modeling of fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3905.
Abstract 3907: Invasive properties of GSCs with known IDH1 status into human cerebral organoids
Authors: Munyeshyaka Maxime , Singh Sanjay , Gumin Joy , Yang Jing , Ledbetter Daniel , Lang Frederick
Published: September 2020

Glioblastomas (GBM) are malignant Grade IV tumors whose prognosis for patients remains dismal, with median survival of 14.6 months after the standard therapy. GBMs have a high proliferative index and are highly invasive to normal brain tissue. GBMs characteristically exhibit areas of necrosis and recruitment of abnormal blood vessels. The most common form of GBMs are primary GBMs (~95%), with no prior clinical history and have poor clinical outcome. Secondary GBMs arise from lower-grade gliomas and are associated with IDH1 mutation (R132H/C/G/S). Methylation profiling identified a subtype of GBMs called glioma-CpG island methylator phenotype (G-CIMP+ve) GBMs, and this subtype yields significant survival benefit compared to non-G-CIMP+ve GBMs and is mostly associated with IDH1 mutant GBMs. GBM biology is traditionally investigated by utilizing patient-derived tumor cells (e.g GBM stem-like cells “GSCs”) in vitro and in vivo as orthotopic xenografts in immunocompromised mouse models. However, these systems lack normal human neural tissue, and therefore the contribution of the normal human neural microenvironment is unable to be studied using these systems. This shortcoming can be addressed by utilizing human cerebral organoids (COs). Cerebral organoids are derived from induced pluripotent stem cells and can serve as in vitro model systems to recapitulate the human brain microenvironment where multiple neural cell types are represented. A recent study showed that GSCs invade and proliferate in human cerebral organoids, and these tumors phenocopy parental tumors. Here, we hypothesized that the invasiveness of GSCs within COs is dictated by IDH1 status, where IDH1 wild-type are more aggressive than IDH1 mutant GSCs. To test our hypothesis, we utilized 45 day-old COs and three GSCs lines (2 IDH1 wild-type and 1 IDH1 mutant) for in vitro invasion and proliferation assays. These GSCs stably express either green or red fluorescent proteins and can be used as surrogates to identify GSCs within COs. Upon co-incubation for various time points, these GSC bearing COs were fixed, embedded, sectioned, and imaged with a confocal microscope. Our preliminary results show that GSCs with wild-type IDH1 appear to be more aggressive within COs. With further research studies, the use of COs will elucidate the contribution of the normal brain microenvironment to the growth and invasiveness of GBMs.

Citation Format: Maxime Munyeshyaka, Sanjay Singh, Joy Gumin, Jing Yang, Daniel Ledbetter, Frederick Lang. Invasive properties of GSCs with known IDH1 status into human cerebral organoids [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3907.
Abstract 3954: Validation of the extracellular matrix protein fibulin-3 as a molecular target in malignant pleural mesothelioma
Authors: Roshini Arivazhagan , Goparaju Chandra , Nandhu Mohan S. , Longo Sharon L. , Longo John A. , Chou Joan , Middleton Frank A. , Pass Harvey I. , Viapiano Mariano S.
Published: September 2020

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor of the pleura with poor prognosis and limited therapeutic options. Fibulin-3 (gene EFEMP1) is an extracellular matrix protein found in the parenchyma and pleural effusions of MPM, which has been proposed as prognostic biomarker complementing diagnostic biomarkers -such as mesothelin- for this cancer type. However, the functions and potential mechanisms of fibulin-3 in MPM remain completely unknown. To further define the relevance of fibulin-3 in MPM we performed gain- and loss-of-function experiments by respectively overexpressing fibulin-3 in normal mesothelial cells or knocking down its expression in MPM cells, followed by evaluation of cell viability, colony formation, invasion and chemoresistance. We also evaluated changes in gene expression and signaling mechanisms after fibulin-3 downregulation. Furthermore, a novel anti-fibulin-3 antibody was developed and tested for its ability to recognize fibulin-3 in mesothelioma, inhibit pro-tumoral signaling, and disrupt tumor growth in orthotopic MPM models. Fibulin-3 downregulation decreased viability, clonogenic capacity, and invasiveness of MPM cell lines, whereas overexpression of this protein increased the same phenotypic traits in normal mesothelial cells. At the molecular level, fibulin-3 regulated the activation of PI3K/Akt and NFkB signaling and correlated with a gene expression signature required for cell adhesion and motility, matching its cellular effects. Loco-regional delivery of anti-fibulin-3 had a marked cytostatic effect and significantly increased median survival and the number of long-term surviving animals with stable disease. Our work reveals that fibulin-3 is a relevant therapeutic target in mesothelioma, adding to its relevance as prognostic biomarker and encouraging further development of anti-fibulin-3 targeted therapies for this cancer type.

Citation Format: Arivazhagan Roshini, Chandra Goparaju, Mohan S. Nandhu, Sharon L. Longo, John A. Longo, Joan Chou, Frank A. Middleton, Harvey I. Pass, Mariano S. Viapiano. Validation of the extracellular matrix protein fibulin-3 as a molecular target in malignant pleural mesothelioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3954.
Abstract 3970: Tumor-associated macrophage, angiogenesis and lymphangiogenesis predicts prognosis in patients with non-small cell lung cancer
Authors: Hwang Ilseon , Park Keun Uk , Jung Eun Hee , Ryoo Hun-Mo , Ylaya Kris , Chung Joon-Yong , Hewitt Stephen M.
Published: September 2020

Background: The tumor microenvironment (TME) is a critical player in tumor progression, metastasis and therapy outcome. Tumor-associated macrophages (TAMs) are a well-recognized core element of the TME, and are generally characterized as M2-like macrophages. TAMs are believed to contribute to tumor progression, but the mechanism behind this remains unclear. We aimed to investigate the clinical value, angiogenic and lymphangiogenic significance of TAMs in non-small cell lung cancer (NSCLC).

Methods: We assessed CD68, CD163, VEGF-A, and VEGF-C expression in 349 patients with NSCLC by combined immunohistochemistry and digital image analysis. Subsequently, the potential association between M2 TAMs and angiogenetic VEGF-A and/or lymphangiogenetic VEGF-C was evaluated for its prognostic value. Furthermore, the effects of M2 TAMs on angiogenesis and lymphangiogenesis were explored in vitro co-culture system.

Results: Elevated M2 (CD163+/CD68+) ratio was significantly associated with poor overall survival (p=0.023). Dual high expression of M2 ratio and VEGF-C was correlated with worse overall survival (p=0.033). Furthermore, CD68 and CD163 expression are positively associated with VEGF-A and/or VEGF-C expression. Multivariate analysis revealed that M2 ratio was an independent predictor of poor survival [HR (95% CI) = 1.53 (1.01-2.33), p=0.046]. Combined M2+/VEGF-C+ expression was also an independent predictor of poor survival [HR (95% CI) = 2.01 (1.28-3.16), p=0.003]. Notably, we confirmed that M2 macrophages enhanced the protein and mRNA expression of both VEGF-A and VEGF-C significantly, while M1 macrophages induced only mRNA expression of VEGF-A significantly in A549 cells.

Conclusions: This study suggests that TAMs are significantly associated with angiogenesis or lymphangiogenesis in the progression of NSCLC. Furthermore, elevated M2 ratio and combined M2 and VEGF-C high expression are strong indicators of poor prognosis in patients with NSCLC.

Citation Format: Ilseon Hwang, Keun Uk Park, Eun Hee Jung, Hun-Mo Ryoo, Kris Ylaya, Joon-Yong Chung, Stephen M. Hewitt. Tumor-associated macrophage, angiogenesis and lymphangiogenesis predicts prognosis in patients with non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3970.
Abstract 4018: Interaction site elucidation of the Rac1/Cdc42 inhibitor, MBQ-167
Authors: Medina Julia I. , Bayro Marvin , Hernandez Eliud , Vlaar Cornelis , Gonzalez Ricardo , Dharmawardhane Suranganie
Published: September 2020

Metastasis continues to be the primary cause of cancer-related death. Therefore, we focus on designing small molecules inhibitors to block metastasis. The Rho GTPase family members Rac1, and Cdc42 are critical regulators of cancer cell migration and invasion, and thus, metastasis. Rac1 is activated by Guanine Nucleotide Exchange Factors (GEFs), which promote the exchange of GDP to GTP. In most cancers, the homologous Rac1 and Cdc42 are hyperactivated due to the overexpression of GEFs. Therefore, we designed inhibitors to block the activation and interaction of Rac1 and Cdc42 with their GEFs. Our lead compound MBQ-167, is a dual inhibitor of Rac1 and Cdc42 with IC50s of 103 nM and 78 nM, respectively. MBQ-167 inhibited the viability and migration of metastatic cancer cells without affecting non-metastatic or non-cancer cells. Moreover, MBQ-167 reduced mammary tumor growth and metastasis of HER2 positive and triple-negative breast cancer cells in immunocompromised mice by ~ 90%. To further elucidate the mechanism of action of MBQ-167, we tested the hypothesis that MBQ-167 blocks the interaction of specific Rac1/Cdc42 GEFs by interaction with particular amino acid residues in the switch I and II homologous regions of Rac1 and Cdc42 proteins. Therefore, to achieve the objective of elucidating the interaction site of MBQ-167 on Rac1 and Cdc42, we used in silico analysis and Nuclear Magnetic Resonance (NMR) of MBQ-167 with Rac1 in a GDP bound state. These results were biochemically validated by pulldown assays using a G15A nucleotide-free mutant predicted to interact tightly with Rac1/Cdc42 GEFs. In silico, using Autodock Tools, MBQ-167 was docked to the GEF binding domain of Rac1 and Cdc42 with low binding energy, indicating a tight interaction. For the NMR structural analyses, Rac1 was isotopically labeled with 15N and titrated with several concentrations of MBQ-167, at ratios of 1:1, 1:2, 1:3, and 1:4. We were able to detect chemical shift perturbations in key residues in the switch I and II regions that interact with Rac1/Cdc42 GEFs and regulate the activity. Moreover, these perturbations were more intense with increasing amounts of MBQ-167, confirming the specificity of the interaction. Therefore, our data signify the direct interaction of MBQ-167 with Rac1, thus validating the role of MBQ-167 as a specific Rac1/Cdc42 inhibitor.

Citation Format: Julia I. Medina, Marvin Bayro, Eliud Hernandez, Cornelis Vlaar, Ricardo Gonzalez, Suranganie Dharmawardhane. Interaction site elucidation of the Rac1/Cdc42 inhibitor, MBQ-167 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4018.
Abstract 4027: Metformin treatment inhibits uterine leiomyoma and leiomyosarcoma cells growth and migration in vitro
Authors: Ricci Anamaria R. , Maffazioli Giovana D. , Baracat Edmund C. , Carvalho Kátia C.
Published: September 2020

Leiomyoma (LM) and leiomyosarcoma (LMS) are uterine mesenchymal tumors that present variable clinical and biological behavior. They are associated with infertility, pelvic pain and abnormal uterine bleeding.. LM is a benign tumor commonly affecting women at reproductive age whereas LMS represents 40% of all uterine sarcomas. These tumors do not have specific therapy and their origin is still uncertain. Some researchers believe that degenerated LM may to turn to LMS. FOXO3a, a transcriptional factor, has been associated to regulation of several cellular functions as well as tumors development. Previously, we detected that FOXO3a gene and protein expression present different expression profiles in LM and LMS. In this current study, our aim was to investigate the in vitro effects of metformin and genistein, both inhibitors of AKT-mediated FOXO3a phosphorylation, treatment in LM and LMS cells behavior. LM (THESCs CRL-4003) and LMS (SK-UT-1) cell lines were grown in specific mediums and conditions (ATCC recommendations), and treated with different drugs concentrations (0.1 - 200 uM and 0.1 - 200 nM, for metformin and genistein, respectively) at several time points (0, 24, 48, 72, 96 and 120 h). After treatment, cells were evaluated for gene and protein expression, cell proliferation, migration and apoptosis. Gene expression was assessed by quantitative Real Time PCR and proteins were evaluated by immunocitochemical analysis. Genistein depicted high toxicity for the cells, due to its vehicle (DMSO), in all tested dose and times. Metformin presented higher efficiency in cell proliferation and migration inhibition for both cell lines, as well as in the apoptosis induction. Prominent effects were observed in LMS cells after treatment with 25 uM for 72h. Gene expression analysis showed a slight decrease in FOXO3a expression after treatment with these drugs. Our results suggest that treatment with metformin induces the FOXO3a function restoration in the cells, by inhibition of the AKT/PI3K pathway, and can represent a future alternative target therapy for these neoplasias.

Citation Format: Anamaria R. Ricci, Giovana D. Maffazioli, Edmund C. Baracat, Kátia C. Carvalho. Metformin treatment inhibits uterine leiomyoma and leiomyosarcoma cells growth and migration in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4027.