bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3788: CircHMGCS1 interacts with RNA binding protein HuR and maintains stem-like cells in gliomas
Authors: He Jiehua , Huang Zuoyu , Yin Dong
Published: September 2020

Malignant Glioma account for the largest class of primary brain tumor, and its high malignant degree, therapy resistance and high recurrence rate are widely accepted to be caused by existence of glioma stem-like cells (GSCs). But the mechanisms underlying GSCs maintain are not completely clear. Recent evidences have shown that circular RNAs (circRNAs) are frequently dysregulated and play paramount roles in various cancers. circRNAs are abundant in central nervous system (CNS); however, few studies describe the clinical significance and role of circRNAs in gliomas, In our preliminary study, we used a vitro differentiation model upon inducing human neural progenitor cells (hNPCs) to mature glia cells, and identified a circRNA, circHMGCS1 (hsa_circ_0072391), which was highly expressed in hNPCs but was gradually downregulated in mature glia cells. In this study, we found that circHMGCS1 was inversely elevated in gliomas, and was positively correlated with malignant degree (III+IV vs I +II, P<0.05). Next, we verified that circHMGCS1 enhanced formation of neurosphere, and leaded to the resistance to TMZ treatment in vitro. Furthermore, we proved that circHMGCS1 could maintain the protein level of stemness relative transcription factor SOX2. RNA binding protein HuR, which was reported to stabilize the SOX2 mRNA, could be pulled down by circHMGCS1 in our ChIRP-MS assay. Inhibition of circHMGCS1 could partly decrease the binding of HuR with SOX2 mRNA 3’UTR. Finally, silencing of circHMGCS1 significantly inhibited formation of xenograft brain tumors and increased mice overal survival time in vivo. Taken together, our findings suggest that circHMGCS1 is a critical player in maintaining the stemness of GSCs via stablilized SOX2 by strengthening its interaction with RNA binding protein HuR. Therefore, we suggest that circHMGCS1 may serve as a new biomarker and therapeutic target for treatment of gliomas.

Citation Format: Jiehua He, Zuoyu Huang, Dong Yin. CircHMGCS1 interacts with RNA binding protein HuR and maintains stem-like cells in gliomas [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 3788.
Abstract 3795: Investigate the mechanism of actions of flavonoid dimers in reversing doxorubicin resistance in breast cancer stem cells
Authors: Sun Gege , Wong Iris L. , Cui Jiahua , Liu Zhen , Chan Bill T. , Chow Larry M.
Published: September 2020

Drug resistance, radioresistance and metastasis are the biggest hurdle in effective cancer treatment. Recent studies suggest that these problems might be caused by the presence of cancer stem cells (CSCs). CSCs are a small group of cells which can self-renew, remain undifferentiated, resist to cancer drugs and radiotherapy. It can be predicted that selective killing of CSCs may lead to tutor regression and may be a new therapeutic solution. Naturally occurring flavonoids are weakly active in inhibiting ABC transporter leading to drug accumulation in cancer cells. We have been working on the design, synthesis and characterisation of novel flavonoid dimers that can be used to reverse drug resistance in cancer cells. We recently discovered that a new class of triazole-linked flavonoid dimers 13_9 and 15_2 were effective in reversing drug resistance in CD44+CD24-/low MCF7 breast cancer stem cells at an effective concentration of less than 32 nM. Our studies suggested that a novel mechanism was involved which is independent of ABC transporters. To determine the mechanism behind, we have designed and synthesised a photoactivatable derivative of 13_9 (13_9X2) to identify the protein target(s). 13_9X2 contains a photoactivating functional group which can capture the target protein by forming a covalent bond under UV irradiation and a clickable functional group which can be used for pull-down and further identification using mass spectrometry. One candidate target is peroxiredoxin 1 (PRDX-1) which is a reactive oxygen species (ROS) scavenger. We hypothesise that CSCs are resistant to doxorubicin-induced ROS damage by overexpressing PRDX-1. By binding and disrupting PRDX-1, 13_9 and 15_2 reduce ROS scavenging ability of PRDX-1, resulting in the reversal of drug resistance in CSCs. This work provides new insights into the mechanisms of cell signalling regulation of cancer cell resistance in breast CSCs and provides a better understanding of how flavonoid dimers reverse drug resistance in CSCs. Additionally, this work contributes to the development of new anticancer drugs which can sensitize CSCs to current chemotherapy.

Citation Format: Gege Sun, Iris L. Wong, Jiahua Cui, Zhen Liu, Bill T. Chan, Larry M. Chow. Investigate the mechanism of actions of flavonoid dimers in reversing doxorubicin resistance in breast cancer stem cells [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 3795.
Abstract 3813: Activation of EGFR induces loss of primary cilia in oral mucosa carcinogenesis
Authors: yin Fengg , Wei Zihao , Li Jing , Xie Liang , Chen Qianming
Published: September 2020

Objective: Primary cilia are evolutionally conserved cell structures involving multiple cell functions by sensing extracellular signals. Loss of primary cilia is frequently observed in various cancer types, suggesting that the absence of this organelle may promote tumorigenesis. However, little is known about the role of primary cilia in oral premalignant lesions and oral squamous cell carcinoma (OSCC). Materials and Methods: Immunofluosense staining was carried out to detect the incidence of primary cilia in every stages of oral carcinogenesis from normal to OSCC. Immunohistochemical analysis was used for validating the correlation between the ciliary incidence and the expression level of EGFR in the oral carcinogenesis. Furthermore, the change of primary cilia was detected by activating and inhibiting the EGFR pathway activity in vitro.

Results: A significant decrease in the percentage of ciliated cells was found on both oral leukoplakia (OLK), as well as OSCC tissues, compared to normal oral mucosa epithelium, and this loss of cilia was independent of the increased proliferative index. A parallel increase of EGFR activity was observed in OLK and OSCC tissues. In addition, EGF stimulation induced obvious primary cilia absorption, whereas inhibition of EGFR by gefitinib significantly restored cilia formation in vitro. We further found that the activation of EGFR resulted in the phosphorylation of AURKA, a pivotal kinase promoting disassembly of primary cilia.

Conclusion: EGFR-AURKA pathway might be a critical regulator of ciliary loss in the carcinogenesis of oral mucosa. This may provide insight into the development of new therapeutic strategies to prevent oral premalignant lesions from becoming malignant.

Citation Format: Fengg yin, Zihao Wei, Jing Li, Liang Xie, Qianming Chen. Activation of EGFR induces loss of primary cilia in oral mucosa carcinogenesis [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 3813.
Abstract 3826: The zinc transporters, ZnT5 and ZnT6, in breast cancer
Authors: Iwabuchi Erina , Miki Yasuhiro , Ishida Takanori , Sasano Hironobu
Published: September 2020

The zinc levels in breast cancer tissue were reported to be higher than those in normal tissue. In addition, the expression levels of zinc transporters, including ZnT5 and ZnT6, in breast cancer were reported to be higher than those in normal breast tissue. ZnT5 and ZnT6 have been also reported to contribute to the formation of heterodimers and be involved in several different biological functions. Among those, autotaxin, matrix metalloproteinase 9, and carbonic anhydrase IX, which have crucial roles in tumor formation, invasion, and metastasis, were reported to require ZnT5–ZnT6 heterodimers for their activation and secretion in the chicken DT40 cells. However, the functions of ZnT5 and ZnT6 heterodimers has remained virtually unknown in breast cancer. Therefore, we first immunolocalized ZnT5 and ZnT6 in pathology specimens of breast cancer. We examined 88 breast cancer specimens in this study. We then analyzed the interaction between ZnT5 and ZnT6 using proximity ligation assay (PLA) in breast cancer tissues and further evaluated cell proliferation and migration in ZnT5 knockdown MCF-7 and T-47D cells using siRNA. Immunohistochemical analysis revealed that the number of ZnT5-positive breast cancer cells was significantly higher in HER2-positive patients and the number of ZnT6-positive breast cancer cells was significantly higher in patients with low histological grade and high ER and PgR expressions. The number of ZnT5 and ZnT6 double-positive breast cancer cells tended to be associated with low stage (p = 0.0577). In addition, ZnT5 and ZnT6 interaction was detected using PLA through high expression of both ZnT5 and ZnT6 in breast cancer tissues. ZnT5 knockdown exerted no effect on cell proliferation in the presence of 100 µM ZnCl2 in MCF-7 and T-47D cells. The 100 µM ZnCl2 treatment inhibited cell migration in MCF-7 and T-47D cells but ZnT5 knockdown promoted cell migration. To the best of our knowledge, this is the first study demonstrating the expressions of ZnT5 and ZnT6 in breast cancer. The results of this study also indicated that both ZnT5 and ZnT6 could be involved in less aggressive subtypes of breast cancer possibly through inhibition of cell migration. Further studies are required to clarify the clinical significance of their interactions in patients with breast cancer.

Citation Format: Erina Iwabuchi, Yasuhiro Miki, Takanori Ishida, Hironobu Sasano. The zinc transporters, ZnT5 and ZnT6, 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 3826.
Abstract 3828: Tumor promoting effects of sulforaphane on diethylnitrosamine-induced murine hepatocarcinogenesis
Authors: Zheng Jie , Kim Do-Hee , Fang Xi Zhu , Kim Seong Hoon , Saeidi Soma , Kim Su-Jung , Surh Young-Joon
Published: September 2020

Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor involved in protection against initiation of carcinogenesis in normal cells. Notably, recent studies have demonstrated that mutation of the gene encoding this transcription factor causes its overactivation in various types of human malignancies. The aberrant activation of NRF2 is considered to accelerate the proliferation and progression of cancer cells. However, the exact function of NRF2 in the stage of pre-malignancy remains still largely unresolved. The differential effects of NRF2 on multi-stage carcinogenesis have raised a concern about the validity of NRF2 activators for chemoprevention. In the present study, a well-known NRF2 activating chemopreventive phytochemical, sulforaphane (SFN), was daily injected intraperitonally (25 mg/kg) for 3 months to male C57BL/6 mice at 6 months after single intraperitoneal administration of a hepatocarcinogen, diethylnitrosamine (DEN). The relative fold changes of liver weight, tumor growth, and the number and the size of hepatomas measured at 9 months after DEN administration were significantly higher in SFN-treated mice than that in vehicle-treated mice. Moreover, the expression of NRF2, its target protein NAD(P)H:quinone oxidoreductase 1 (NQO1), and the cell proliferation marker proliferating cell nuclear antigen (PCNA) and the DNA damage marker gamma-histone 2A family member X (γH2AX) was markedly elevated in SFN-treated mice compared with that in vehicle-treated mice as assessed by the Western blot assay. Immunohistochemistry and Immunofluorescence staining data further verified higher expression of NRF2 and PCNA in SFN-treated mice. These results suggest that once HCC is initiated, SFN stimulates the progression of DEN-induced HCC via activation of NRF2, rather than exerting a cancer chemopreventive effect. RNA sequencing analysis with liver tumors from DEN-treated mice revealed the B-raf V637E mutation (4/7). We speculate that mutation of B-raf proto-oncogene may contribute, at least in part, to activation of oncogenic NRF2 in DEN-induced murine carcinogenesis. The effects of SFN on DEN-induced mutation spectrum is under investigation. This study was supported by the Global Core Research Center (GCRC) Grant (No. 2011-003-0001) from the National Research Foundation (NRF) of Republic of Korea.

Citation Format: Jie Zheng, Do-Hee Kim, Xi Zhu Fang, Seong Hoon Kim, Soma Saeidi, Su-Jung Kim, Young-Joon Surh. Tumor promoting effects of sulforaphane on diethylnitrosamine-induced murine hepatocarcinogenesis [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 3828.
Abstract 3876: Multiplex immunohistochemistry accurately defines the immune compositional change of tumor microenvironment to predict hyperprogressive disease
Authors: Kim Seoree , Chun Sang Hoon , Kim Sang-Yeob , Seo Junyoung , Jung Chan Kwon , Kang Jinhyoung
Published: September 2020

Introduction: To retrospectively assess hyperprogressive disease(HPD) in patients who underwent immune checkpoint blockade therapy and explore the heterogenous nature of tumor microenvironment of hyperprogressive patients using multiplex immunohistochemistry.

Method: We retrospectively reviewed medical records of non-small cell lung cancer patients (n=243) treated with anti-PD-1 or anti-PD-L1 monotherapy at 5 institutes of St. Mary's Hospitals between January 2014 and May 2018. HPD was defined as a tumor growth kinetics ratio (TGKr) exceeding two during anti-PD-1 / PD-L1 therapy and a time-to-failure (TTF) of less than 2 months. We also analyzed the association of Neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR) and CRP-albumin ratio (CAR) with HPD. To evaluate the difference of immune composition in TME by multiplex IHC(mIHC). Panel 1/2 composed of T cell markers and co-inhibitory signal markers. Panel 3/4 examined macrophages, NK cells and dendritic cells.

Results: Overall, 231 patients were included. Overall, 26 patients (11.3%) met the criteria for hyperprogression. Multiplex IHX data quantitated immune subset number present intra-tumor vs the tumor stroma. there was a difference in immune cell composition between the HPD, poor responders (PRs) and good responders (GRs). High levels of CD4 + Teff cells, FOXP3 Treg cells were associated with HPD(p < 0.052, <0.017) and CD4 to CD45RO ratio indicated a potential predictive value to develop HPD(p < 0.042). Key observation was that there was a tendency to M2 polarization in patients with HPD when the macrophage M1/2 markers of CD14, CD68 and CD163 were observed. And the myeloid markers, such as CD11c, expressed by CAFs, tend to increase in the intra-tumor lesion of the HPD group (p < 0.501).

Conclusion: This study was important in investigating the HPD-related immune prognostic factors in NSCLC patients in korea. According to these data, HPD is a unique biologic process distinct from a non HPD-PR. Multiplex IHC can be used not only to predict HPD, but also to understand the dynamicity and the complexed immune compositional change within TME. Furthermore, based on these findings, we are undergoing study the TME by serially collected pre- and post-treatment tissue samples, it may be helpful to shed light on the mechanisms of HPD.

Citation Format: Seoree Kim, Sang Hoon Chun, Sang-Yeob Kim, Junyoung Seo, Chan Kwon Jung, Jinhyoung Kang. Multiplex immunohistochemistry accurately defines the immune compositional change of tumor microenvironment to predict hyperprogressive disease [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 3876.
Abstract 3877: A unique layered 3D stromal tissue model for in vitro evaluation of immunologic agents and immune cell infiltration into microenviroment
Authors: Morimura Rii , Nada Isana , Tsukamoto Kei , Shinozaki Eiji , kitano shiro
Published: September 2020

Background

The modulation of the immune system is a proven key target for cancer therapy, however, the complex interplay between immune cells and cancer cells in cancer microenvironment remains only partially understood. In addition, the currently available in vitro cancer microenvironment models are not designed to study the immunologic agents and a role of immune cells. Here, we developed a unique layered 3D stromal tissue model comprising immune, stromal and cancer cells.

Method

Layered 3D stromal tissue model was constructed by culturing fibroblasts and endothelial cells coated with extracellular matrix (ECM) and natural polysaccharide. Morphological analysis of the layered 3D stromal tissues and co-cultured tumor were evaluated by HE stain, immunohistochemistry and immunofluorescence (IF). For cytotoxicity assay, cancer cell lines were seeded on or in the 3D stromal tissue and incubated with peripheral blood mononuclear cell (PBMC) and Programmed cell death 1 (PD-1) blockade with Nivolumab. Remaining cancer cells post drug treatment were quantified by IF and imaging analysis.

Result

The co-cultured stromal tissues were multi-layered (approximately 20 layers), had microvascular formation. Hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) derived from fibroblast cells played important roles in tube formation. Moreover, the position of cancer cells in 3D stromal tissue was manipulated easily. PBMC infiltration into 3D stromal tissue and the interaction between PBMC and cancer cells were visualized. High level of programmed cell death ligand 1 (PD-L1) expression on cancer cells correlated with a higher response to PD-1 blockade in vitro as well as clinical trials.

Conclusion

We present a unique layered 3D cell culture system based on stromal/cancer/immune cells to study immunologic agents and immune cell infiltration in cancer microenvironment.

Our 3D model has the ability to reflect in vivo conditions closer than 2D model and provide a tool for in vitro tumor infiltrating lymphocyte (TIL) or immunologic agent testing.

Citation Format: Rii Morimura, Isana Nada, Kei Tsukamoto, Eiji Shinozaki, shiro kitano. A unique layered 3D stromal tissue model for in vitro evaluation of immunologic agents and immune cell infiltration into microenviroment [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 3877.
Abstract 3882: YAP1 and WWTR1 (TAZ) positively regulate PAX3-FOXO1 transcriptional programming in fusion-positive rhabdomyosarcoma
Authors: Rashid Tooba , Burgess Breanne A. , Linardic Corinne M. , Deel Michael D.
Published: September 2020

Although more than 20 years have passed since the discovery that fusion-positive rhabdomyosarcoma (FP-RMS) is driven by the chimeric fusion oncogene PAX3-FOXO1 (P3F), therapeutically tractable components of the P3F tumorigenic program have yet to be uncovered and survival rates remain dismal (5-yr overall survival <50%). YAP1 and TAZ, transcriptional co-activators of the Hippo pathway, are potent oncogenes known to mediate transcriptional addiction in malignancy. Here, we demonstrate that YAP1/TAZ complex with P3F and positively regulate P3F transcriptional activity in FP-RMS. Immunohistochemical (IHC) staining of human tissue microarrays were used to identify the relative abundance of YAP1 and TAZ in FP-RMS tumor samples. Functional interactions of YAP1/TAZ and P3F were investigated using P3F reporters, immunoblots, and co-immunoprecipitation (co-IP); while co-IP-coupled mass spectrometry (IP-MS) was used to identify the YAP1/TAZ/P3F interactome. Experiments utilized gain- and loss-of-function vectors expressing control, wild-type YAP1/TAZ, constitutively active YAP1/TAZ (S89A/S127A), or shRNA knockdown. To identify P3F-mediated genes and pathways that are YAP1/TAZ-dependent, we used RNA-Seq and quantitative proteomics via tandem mass tag labeling. We demonstrate via IHC that YAP1/TAZ are highly abundant in FP-RMS, and through functional assays that YAP1/TAZ regulate many FP-RMS cancer phenotypes. Mechanistically, an interaction between YAP/TAZ and P3F was demonstrated via co-IPs for endogenous as well as epitope-tagged proteins. This was confirmed with IP-MS, which also revealed that YAP/TAZ and P3F share an enrichment for co-immunoprecipitated proteins involved in DNA binding and transcriptional regulation. IP-MS also demonstrated that chromatin remodeling complex proteins were enriched with TAZ immunoprecipitation. YAP1/TAZ functionally augment P3F transcriptional activity in reporter assays as well as expression of candidate P3F target genes. An unbiased approach using RNA-Seq and quantitative proteomics demonstrate that YAP1/TAZ positively regulate differential expression of P3F target genes, as well as proteins involved in cell cycle progression and pan-cancer related proteins that are typically up- or down-regulated across multiple malignancy types.In conclusion, we identify a novel complex between YAP1/TAZ and P3F, show YAP1/TAZ are positive regulators of P3F transcriptional activity, and identify the YAP1/TAZ axis as a vulnerability for P3F-transcriptional reprogramming in FP-RMS.

Citation Format: Tooba Rashid, Breanne A. Burgess, Corinne M. Linardic, Michael D. Deel. YAP1 and WWTR1 (TAZ) positively regulate PAX3-FOXO1 transcriptional programming in fusion-positive rhabdomyosarcoma [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 3882.
Abstract 3904: The novel combination of PAC-1 and ONC201 circumvent H3K27M-driven pro-survival gene expression to induce DIPG cell death
Authors: Parkhurst Ashlyn , Park Youngran , Arnold Antje , Koldobskiy Michael , Eberhart Charles , Raabe Eric , Rubens Jeffery
Published: September 2020

Diffuse Intrinsic Pontine Gliomas (DIPGs) are universally fatal tumors, resistant to the cytotoxic effects of all known therapies. We aim to better understand the mechanisms contributing to this resistance to design new combination therapies that will improve DIPG survival. DIPG's aggressive phenotype is driven by the H3K27M oncohistone mutation that leads to global genomic DNA hypomethylation and abnormal gene expression. To identify patterns of gene expression driven by the H3K27M mutation, we introduced the H3K27M mutation into murine neural stem cells and performed whole genome bisulfite sequencing (WGBS) to evaluate for genes with the greatest differential methylation in their promoter region. These studies identified an abundance of pro-survival genes such a BCL-2, BCL-xL, MCL-1, Caspase-3, and XIAP. Western blot confirmed elevated expression of these pro-survival factors and RNA-Seq of primary human DIPG confirmed increased expression compared to normal brain. PAC-1 is a compound that cleaves protective zinc bonds to activate procaspase-3, directly inducing cell death. ONC201 is a small molecule that inhibits the anti-apoptotic protein XIAP, which prevents activated caspase-3 from translocating to the nucleus to induce apoptosis. We hypothesized that PAC-1 combined with ONC201 will circumvent these protective abnormalities in the DIPG apoptotic signaling cascade to induce tumor cell death. PAC-1 induces high rates of apoptosis in DIPG (25µM concentrations increases the percent of cells undergoing apoptosis; cleaved caspase 3 immunofluorescence assay 5% to 30% (t-test p<0.05), ANNEXIN V Cell sorting assay 11% to 40% (t-test p<0.05), and western blot for cPARP.) PAC-1 synergizes with ONC201 to further induce apoptosis in DIPG. Combination therapy decreases the percent of viable cells by >40% compared to DMSO control (ANOVA p<0.05 compared to each medication alone and DMSO control). Combination therapy also increases the induction of cPARP compared to each medication alone (western blot). These studies identified H3K27M-mediated expression of pro-survival genes in DIPG. The novel combination of PAC-1 and ONC201 therapy synergize to circumvent this abnormal apoptotic signaling cascade to induce cell death. Our data support future clinical trials combining PAC-1 with ONC201 to extend survival in DIPG.

Citation Format: Ashlyn Parkhurst, Youngran Park, Antje Arnold, Michael Koldobskiy, Charles Eberhart, Eric Raabe, Jeffery Rubens. The novel combination of PAC-1 and ONC201 circumvent H3K27M-driven pro-survival gene expression to induce DIPG cell death [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 3904.
Abstract 3934: UBXN2A inhibits tumor invasion and metastasis by targeting the mTORC2-VEGF pathway
Authors: Eikanger Morgan , Srinivasan Rekha , Sane Sanam , Rezvani Khosrow
Published: September 2020

Despite advances in treatment regimens, one-third of colorectal cancer (CRC) patients will die from this metastatic disease that can disseminate, or spread, throughout the body. Therefore, understanding the mechanisms underlying the multistep metastatic program of CRC cells is critical for the development of novel therapies that will improve the management of this advanced disease. Our preliminary data indicate that a ubiquitin-like protein, UBXN2A, function as mTORC2 inhibitor in colon cancer cell lines. Our flow cytometry and western blot experiments revealed that induction of UBXN2A with Doxycycline or enhancement of UBXN2A expression with a pharmacological tool, Veratridine, inhibits mTORC2 complex in primary and metastatic colon cancer cells. Based on these results, we hypothesized that increased expression of UBXN2A suppresses the vascular endothelial growth factor, or VEGF protein, which is a key factor in tumor angiogenesis. Our current results indicate that the induction of UBXN2A significantly reduces VEGF protein, a key component in tumor invasion and metastasis. Collectively, our results suggest UBXN2A is a potential key player in intercepting the angiogenesis pathway and downstream regulators of metastasis by suppressing VEGF protein. These findings provide an attractive and promising target for the treatment of metastatic CRC.

Citation Format: Morgan Eikanger, Rekha Srinivasan, Sanam Sane, Khosrow Rezvani. UBXN2A inhibits tumor invasion and metastasis by targeting the mTORC2-VEGF pathway [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 3934.
Abstract 3938: Anti-angiogenic therapy combination with hypoxia-regulating agent leads to improved tumor regression in a murine model or renal carcinoma
Authors: Nafia Imane , Chaibi Assia , Bortolotto Doriane , Rey Christophe , Bessede Alban
Published: September 2020

Background: While antiangiogenic therapies have been shown effective in preclinical and clinical trials, tumors can acquire resistance conferring them the ability to survive and grow under hypoxic conditions, even, induced by antiangiogenic drugs themselves.

Methods: This study investigated the effects of an antiangiogenic strategy and hypoxia-regulating agent, each alone and in combination. Here, we have tested the synergistic activity of the anti-angiogenic agent Sunitinib and the mTOR inhibitor Everolimus in murine syngeneic renal cancer model - renal carcinoma being known as angiogenesis-dependent and hypoxia-driven malignancy. Anti-tumor effects were investigated on tumor growth and survival. Flow cytometry-based immunoprofiling was performed at the peripheral level - blood and spleen - while immunohistofluorescence and quantitative image analysis served to characterize and evaluate tumor immune cell infiltrate, tumor hypoxia, and tumor vascular density/features.

Results: While Sunitinib and more importantly Everolimus led to a significant tumor growth inhibition as single agents, their combination was more effective on tumor regression. At the peripheral level, Sunitinib, but also Everolimus, led each to declines in circulating and splenic myeloid-derived suppressor cells (MDSCs) - depicted as CD11b+/Gr1+, an event that was further optimized upon combination of both agents. In addition, tumor sections analyses showed decreased microvessel density under Sunitinib and combination treatment. Differential modulations of correlative endothelium/pericytes staining and hypoxia degree were also observed upon the different treatments. This study confirms the preclinical efficacy of Sunitinib and a hypoxia regulator in renal tumor model and suggests that limitation of Sunitinib-induced hypoxia through mTOR targeting or with hypoxia regulators represents a suitable strategy to enhance effect of anti-angiogenic agent thereby limiting tumor growth.

Citation Format: Imane Nafia, Assia Chaibi, Doriane Bortolotto, Christophe Rey, Alban Bessede. Anti-angiogenic therapy combination with hypoxia-regulating agent leads to improved tumor regression in a murine model or renal 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 3938.
Abstract 3951: Age-dependent loss of HAPLN1 affects vascular integrity and metastasis in melanoma
Authors: Marino Gloria E. , Almeida Filipe V. , Liu Ying , Alicea Gretchen M. , Fane Mitchell E. , Weeraratna Ashani T.
Published: September 2020

Older melanoma patients (>55 years) have worse prognoses than younger patients (<45 years) independent of other clinical prognostic factors. Further, while younger patients have more lymph node metastases, older patients have more distal metastases which significantly impacts their survival. We have shown that age-dependent loss of a secreted factor from dermal fibroblasts, HAPLN1, contributes to increased distal metastasis in vivo via ECM reorganization. Previous work from our lab has shown that age-dependent loss of HAPLN1 confers fewer lymphatic metastases but more lung metastases, consistent with clinical observations. We have demonstrated that this is due to ECM structural degradation and loss of lymphatic vasculature integrity. However, the biological mechanisms by which dermal ECM structure influences metastatic spread and localization are still largely unknown. Here we report that age-dependent loss of HAPLN1 contributes to increased intratumoral neoangiogenesis and decreased vascular integrity. Using immunohistochemistry, we have shown that tumors from aged mice treated with recombinant HAPLN1 have significantly fewer neoangiogenic blood vessels than untreated aged mice, suggesting that HAPLN1 may signal to inhibit neoangiogenesis. Additionally, the intratumoral blood vessels in aged mice treated with recombinant HAPLN1 have increased levels of the adherens junction protein VE-cadherin consistent with levels seen in the tumor vasculature of young mice. Given the pivotal role of intratumoral vasculature in metastasis, we hypothesize that these changes in vessel number and permeability contribute to the increased incidence of lung metastasis in older patients. Further, our data utilizing two-photon microscopy indicate that aged mice have aberrant collagen structure surrounding intratumoral vessels, but that treatment with recombinant HAPLN1 is sufficient to restore a collagen pattern similar to what is seen in young mice. We have additionally demonstrated using an in vitro impedance-based assay that endothelial cell monolayers have a reduced ability to maintain barrier function in the context of ECM without HAPLN1. Given that dermal ECM structural complexity is essential for maintaining vascular integrity, loss of HAPLN1 seems to contribute to this “leaky” vasculature phenotype. In all, these data may provide clues as to why young and aged patients exhibit differential routes of metastases via lymphatic vs. hematogenous dissemination.

Citation Format: Gloria E. Marino, Filipe V. Almeida, Ying Liu, Gretchen M. Alicea, Mitchell E. Fane, Ashani T. Weeraratna. Age-dependent loss of HAPLN1 affects vascular integrity and metastasis in melanoma [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 3951.
Abstract 3961: Influence of hyaluronic acid in tumor microenvironment on glioblastoma (GBM) invasive behavior
Authors: Solomon Itay , Sohrabi Alireza , Seidlits Stephanie K.
Published: September 2020

Glioblastoma invasion is mediated by distinct interactions between tumor cells and the extracellular matrix. Engineered biomaterial platforms allow for systemic examination of the influence of the microenvironment HA content on GBM cell activity. In this study, high molecular weight hyaluronic acid (700 kDa) was photopolymerized within three-dimensional (3D) polyethylene-glycol (PEG) hydrogels and patient-derived, primary GBM cell migration in the HA-hydrogels was investigated. Hydrogel polymerization conditions were tuned to achieve orthogonal control of HA content in a series of hydrogels (0.1 - 0.75 wt% HA) with conserved poroelastic properties (i.e. storage moduli and diffusivity). GBM cell migration was strongly influenced by microenvironment HA content. Knockdown of HA-dependent cell surface receptors (CD44 and RHAMM) modulated GBM cell activity. Our results demonstrate that alterations in microenvironment HA content and inhibition of HA-dependent signaling produced distinct molecular and phenotypic differences in GBM migratory behavior. These results demonstrate the differential effect of tumor microenvironment HA content on the invasive phenotype of GBM in a physiologically relevant 3D culture environment.

Citation Format: Itay Solomon, Alireza Sohrabi, Stephanie K. Seidlits. Influence of hyaluronic acid in tumor microenvironment on glioblastoma (GBM) invasive behavior [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 3961.
Abstract 3962: Differential extracellular matrix remodeling induced by VEGF-A and TGF-β activated fibroblasts
Authors: Nuñez Muriel A. , Cerda-Infante Javier , Nualart Francisco , Cerda Gustavo , Yussef Isabel M. , Montecinos Viviana P.
Published: September 2020

Introduction: Tumor progression is modulated by the presence within the tumor microenvironment of activated fibroblasts called cancer-associated fibroblasts (CAFs). CAFs exhibit an increase in the production of collagen (COL), tenascin C (TNC) and fibronectin (FN), resulting in a remodeling of the extracellular matrix (ECM) within the tumor mass. CAFs-derived ECM guides the cancer cells to migrate directionally increasing metastasis. TGF-β and VEGF-A are highly express in tumors and promote progression, however, their role in ECM remodeling is poorly understood. This study seeks to analyze the ECM protein remodeling mediated by TGF- β o VEGF-A activated fibroblasts.

Materials and methods: In vitro and in vivo ECM protein remodeling mediated by TGF- β o VEGF-A activated fibroblasts was analyzed through expression of TNC, FN and COL using immunofluorescence and second harmonic generation (SHG); alignment and thickness of the fibers; Organoleptic invasion and 2D migration assay were conducted to study the effect of the stiffness of collagen gels generated by VEGF-A and TGF-β activated fibroblasts in the migration/invasion of tumor cells.

Results and discussion: ECM derived from VEGF-A and TGF-β activated fibroblasts have differential ECM remodeling patterns, including differences in the secretion of TNC, FN, and COL; alignment and thickness of the fibers, and migration/invasion rate. These changes results in a differential stiffness of the ECM, which would alter the directional migration of cancer cells within tumor microenvironment.

Citation Format: Muriel A. Nuñez, Javier Cerda-Infante, Francisco Nualart, Gustavo Cerda, Isabel M. Yussef, Viviana P. Montecinos. Differential extracellular matrix remodeling induced by VEGF-A and TGF-β activated fibroblasts [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 3962.
Abstract 3963: Physical confinement induces malignant transformation in mammary epithelial cells
Authors: Lu Yen-Chun
Published: September 2020

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.

Citation Format: Yen-Chun Lu. Physical confinement induces malignant transformation in mammary epithelial cells [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 3963.
Abstract 4017: Biochemical and biophysical characterization of a covalent inhibitor of Src kinase
Authors: Gurbani Deepak , Westover Kenneth , Bera Asim
Published: September 2020

Background: c-Src was among the first oncogenes discovered. It encodes a non-receptor tyrosine kinase that regulates many cancer-related cellular processes including mitogenesis, angiogenesis, adhesion, invasion, migration, and survival. Src activity drives malignant phenotypes in hematologic and solid cancers including breast, prostate, lung, colorectal, and pancreatic cancer. However, no specific Src-directed targeted therapies are in clinical use. Various Src inhibitors such as dasatinib, bosutinib and others have been tested in human trials, but with variable outcomes including dose-limiting toxicity, which is due to the multi-targeted nature of these compounds, which also hit Src, SFKs, BCR-ABL, c-KIT, PDGFR, c-FMS, EPHA2 and others. Improved selective Src inhibitors may overcome these challenges to enable Src-directed therapies. Engineering selectivity into Src inhibitors is challenging because of the high degree of sequence homology between Src family members and other receptor tyrosine kinases. The selective, covalent Src inhibitor DGY-06-116, which targets a relatively unusual targetable cysteine near the active site in the p loop, was recently reported. Here, we further characterize DGY-06-116.

Methods: We characterized the inhibitory activity DGY-06-116 using an enzymatic assay and surface plasmon resonance (SPR). We also determined the co-crystal structure of DGY-06-116 bound to Src.

Results: DGY-06-116 showed a substantial improvement over both SM171 and bosutinib for inhibition of Src kinase activity: SM1-71 (IC50, 26.6 nM); bosutinib (IC50, 9.5 nM); DGY-06-116 (IC50, 2.59 nM). Determination of kinact using SPR showed a value of 5.7 x 10-7 (s-1) for DGY-06-116, slower than FDA-approved neratinib (2 x 10-3 s-1) and afatinib (1 x 10-3 s-1). A 2.15 Å resolution co-crystal structure of DGY-06-116 bound to Src (PDB ID: 6E6E) showed a covalent bond with Cys-280 and bending of the p-loop relative to other Src-inhibitor structures.

Conclusions: DGY-06-116 demonstrates a slow inactivation rate that likely reflects the need for repositioning of the p-loop for a covalent bond to occur. Src inhibitors based on DGY-06-116 may benefit from optimization of p-loop, inhibitor interactions.

Citation Format: Deepak Gurbani, Kenneth Westover, Asim Bera. Biochemical and biophysical characterization of a covalent inhibitor of Src kinase [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 4017.
Abstract 4021: Assessing the sensitivity of LSF inhibitors against liver cancer
Authors: Pokharel Niranjana , Kavouris John , Biagi Jessica , Hansen Ulla , Schaus Scott E.
Published: September 2020

Introduction: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the second leading cause of cancer mortality. The transcription factor Late SV40 Factor (LSF) functions as an oncogene in HCC, making it a potential protein target for HCC therapy. LSF overexpression correlates with pathogenesis of liver, colorectal and pancreatic cancers, for which there are limited molecularly targeted therapy options. A library of dihydroquinolinones, termed Factor Quinolinone Inhibitors (FQIs), inhibits LSF-DNA binding and specific LSF-protein interactions in in vitro and in cellular assays. The initial lead compound FQI1 causes dramatic mitotic defects in HCC cell lines but has no toxic consequences on immortalized human hepatocytes or primary mouse hepatocytes. Additionally, FQI1 has proven efficacious in endogenous HCC mouse models, with no evidence of associated toxicity.

Methods: A series of dihydroquinolinone compounds were synthesized and tested for potency in two HCC cell lines, Huh7 and SNU423, by a cell proliferation assay. The FQI analogs, FQI34, N-oxide FQI34 and FQI37, were separated by chiral chromatography to the corresponding R and S enantiomers. Direct target engagement of the three lead compounds, FQI1, FQI34 and FQI37, is shown with cellular thermal stability assays on Huh7 cells.

Results: More than 20 compounds were synthesized and characterized. Among them, FQI37 showed the most potent activity (GI50 = 70 nM) against Huh7 HCC cells. Structure-activity-relationship studies suggest that the amide portion of quinolinone core is important for optimal activity. Growth inhibition assays revealed enantiomeric specificity; the (S)-enantiomers are more potent than the (R)-compounds and the racemate. The cellular thermal shift assay in Huh7 cells demonstrated the direct target binding of FQIs to LSF in cells at micromolar concentrations. Growth inhibition assays also identified colorectal cancer and pancreatic cancer lines to be sensitive to the dihydroquinolinones treatment.

Conclusions: Aryl-dihydroquinolinones are promising small molecule chemotherapies for LSF-driven cancers such as HCC, colorectal cancer, and pancreatic cancer.

Citation Format: Niranjana Pokharel, John Kavouris, Jessica Biagi, Ulla Hansen, Scott E. Schaus. Assessing the sensitivity of LSF inhibitors against liver 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 4021.
Abstract 4024: NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay
Authors: Shomali Maysoun , Hilairet Sandrine , Duhamel Marianne , Nardi Frederico , Bertrand Thomas , Penarier Geraldine , Debussche Laurent , Bouaboula Monsif
Published: September 2020

Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD) biosynthesis. As NAMPT inhibitors (NAMPTi) have been shown to be substrates of NAMPT, binding of NAMPTi to NAMPT results in the NAMPTi-phosphoribose adduct (RP) that binds tightly and strongly inhibits the NAMPT enzyme. Structural analyses performed in this study unveiled the proximity of the NAMPTi-RP complex to the phosphoribosyl diphosphate (PRPP)-binding loop within the NAMPT catalytic site. The PRPP-binding loop of NAMPT is subject to conformational flexibility. The PRPP-binding loop domain is oriented to the outer side of the NAMPT dimer and can potentially serve as an antigen-binding site for antibodies. Interestingly, we show that the NAMPTi-RP adduct bound to NAMPT decreases NAMPT capture by an antibody that is directed against the c-terminal 5-phosphoribosyl-1-pyrophosphate-binding loop in NAMPT. This finding was then used to explore cellular NAMPT occupancy. The NAMPTi-RP complex displays a sustained, cellular NAMPT occupancy that correlates with the inhibition of NAMPT activity. Moreover, a good correlation between NAMPT occupancy, NAD decrease, and NAMPTi efficacy was observed in-vivo in a NCI-H82 small cell lung cancer (SCLC) xenograft model. NAMPT-occupancy assay can be used in clinical settings to better define the optimal dose levels and dose regimen for effective NAMPT inhibition.

Citation Format: Maysoun Shomali, Sandrine Hilairet, Marianne Duhamel, Frederico Nardi, Thomas Bertrand, Geraldine Penarier, Laurent Debussche, Monsif Bouaboula. NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay [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 4024.