bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4424: Micro-RNA mediated deregulation of hHB-EGF in ovarian cancer
Authors: Rahman Md. Mustafizur , Sultana Razia , Iwamoto Ryo , Mekada Eisuke
Published: August 2018

Background: Aberrant expression of microRNAs (miRNAs) has been implicated in human carcinogenesis where they function as oncogenes or tumor suppressor genes. Overexpression of HB-EGF, member of the epidermal growth factor (EGF) family, plays pivotal role in ovarian carcinogenesis. However the molecular mechanisms underlying miRNA(s) mediated aberrant function of HB-EGF in ovarian carcinoma remain unresolved. Here, we investigated the miRNA(s) mediated deregulation of HB-EGF in human ovarian carcinogenesis.

Methods: Web-based bioinformatics algorithms were used to identify candidate miRNAs with putative binding sites on target hHB-EGF mRNA. Expression of miRNAs and mRNA were checked by real-time PCR whereas protein expression was investigated by western blotting assay. Luciferase reporter assay was carried out to examine miR-27a target on 3´UTR of hHB-EGF mRNA. Changes in the oncogenic effects of HB-EGF were studied after gain/loss -of-function of the miR-27a using soft agar colony formation assay and 3D collagen culture. In vivo tumor growth potential of miR-27a was measured in BALB/c nude mice.

Results: By using web-based bioinformatics algorithms 11 miRNAs ((miR-27a/b, -29a/b/c, -96, -128, -132, -182,-194, -212) having putative binding sites on the 3′UTR of hHB-EGF mRNA have been selected for further analysis. Among the putative candidates miR-27a showed maximum reciprocal expression with hHB-EGF and thus we selected miR-27a as promising candidate for hHB-EGF regulation. Overexpression of miR-27a significantly inhibited the hHB-EGF expression in SKOV3 cells by almost 50% and miR-27a inhibitors (anti-miR-27a) upregulated the expression of endogenous hHB-EGF mRNA whereas miR-27a overexpression decreased hHB-EGF protein level in MCAS cells suggesting that miR-27a modulate endogenous hHB-EGF expression both at mRNA and protein level. Luciferase reporter assay revealed that miR-27a directly targets 3´UTR of hHB-EGF mRNA at the predicted site. Ectopic expression of miR-27a suppresses in-vitro growth of SKOV3 ovarian cancer cells. In addition, in-vivo tumorigenecity of SKOV3 cells was also inhibited by miR-27a treatment.

Conclusion: The current study has identified novel miR-27a that negatively regulates hHB-EGF expression by directly targeting 3`UTR of its mRNA. This miR-27a might act as oncosupressor in human epithelial ovarian cancer.

Citation Format: Md. Mustafizur Rahman, Razia Sultana, Ryo Iwamoto, Eisuke Mekada. Micro-RNA mediated deregulation of hHB-EGF in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4424.
Abstract 4427: Preparation and mechanism of polyethyleneimine coated miRNA let-7a microspheres
Authors: Lai Honglin , An Weixiao , Zhang Yangyang , Lin Xiukun , Cao Shousong
Published: August 2018

Recent evidence demonstrates that microRNAs (miRNAs) are associated with cancer development and have the potential to be used as anticancer agents. However, the application of miRNAs as clinical cancer drugs has been limited due to their unstability at room temperature. Moreover, miRNAs are negatively charged and excluded by cytomembrane, which has the same polarity. Therefore, miRNAs are difficult to enter cancer cells to exhibit their anticancer effects. In order to solve the problem, we changed the polarity of miRNA let-7a, a member of the second identified miRNA let-7 families, by coating with a bioavailable polymer, polyethylenimine (PEI) using nanotechnology, to alter the charge of miRNA let-7a for positive charge so easily connecting with the anionic residues of cancer cell surfaces. Therefore, miRNA let-7a could easily entered cells through endocytosis. In addition, we performed formaldehyde denaturing SDS-PAGE analysis to determine the stability of miRNA let-7a, and we found the stability of miRNA let-7a has been significantly increased due to microspheres formation. MiRNA let-7a microspheres have showed potent cytotoxicity against a variety of cancer cells including HCT-116, A875, A549, and 4T1 cells with the IC50 as 31.77, 54.34, 44.35, 43.18 pmol/l, respectively by CCK-8 kit analysis. We also found that miRNA let-7a microspheres are able to induce cell apoptosis by promoting the expression of apoptosis-related genes Caspase 3, Caspase 9 and PARP in HCT-116 and A875 cells. Our findings may provide that miRNA let-7a microspheres may be developed as a novel anticancer agents in cancer therapy.

Citation Format: Honglin Lai, Weixiao An, Yangyang Zhang, Xiukun Lin, Shousong Cao. Preparation and mechanism of polyethyleneimine coated miRNA let-7a microspheres [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4427.
Abstract 4440: Differential regulation of p63 isoforms by alternative polyadenylation
Authors: Mohibi Shakur , Zhang Jin , Chen Xinbin
Published: August 2018

p63, a transcription factor belonging to the p53 family, plays vital roles in epidermal development and tumorigenesis. p63 expresses from two different promoters generating two classes of proteins, TAp63 and ΔNp63 that differ in their N-terminus. Moreover, alternative splicing of the TA and ΔN isoforms in the C-terminus creates additional isoforms (α-ϵ); thus generating five TA and five ΔN p63 isoforms. The factors that give rise to this repertoire of p63 isoforms are not very well understood. Alternative polyadenylation (APA) can generate multiple isoforms of a gene with alternative 3' ends based on the polyadenylation site selection during cleavage and polyadenylation of mRNA. APA can give rise to isoforms that differ in the coding region (CDS APA) or that have varying lengths of 3' UTR (3' UTR APA). Recently, PABPN1 protein that binds the 3' poly(A) tail of mRNAs has emerged as a key regulator of APA. Here, we show that PABPN1 differentially regulates p63α and p63γ isoforms by alternative polyadenylation. Specifically, knockdown of PABPN1 increases the levels of p63γ mRNA as well as protein by regulating CDS APA of p63 gene. Moreover, knockdown of PABPN1 also generates p63α mRNAs with varying 3' UTR lengths. Although the mRNA levels of p63α remain unchanged upon PABPN1 KD, p63α protein is significantly decreased, suggesting that PABPN1 also regulates p63α protein translation. Interestingly, we also show that PABPN1 is a p63α target and that p63 transcriptionally regulates PABPN1 by binding to its promoter forming a negative feedback loop. Taken together, our study identifies PABPN1 as a novel regulator of various p63 isoforms by APA and that p63 in turn regulates PABPN1 transcriptionally.

Citation Format: Shakur Mohibi, Jin Zhang, Xinbin Chen. Differential regulation of p63 isoforms by alternative polyadenylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4440.
Abstract 4442: Wilms' tumor 1-associating protein increases cancer cell proliferation and stem-cell like properties through RNA modification
Authors: Park Sae Whan , Oh Young Taek , Park Jong Bae , Kim Jong Heon
Published: August 2018

Purpose: Wilms' tumor 1-associating protein (WTAP) which is known for its role in RNA methylation machinery, is highly upregulated in the necrotic region of glioblastoma multiforme (GBM). The highly heterogenic nature of GBM is associated with the necrotic region within the brain, where many types of cells such as the stromal cell, immune cells, and cancer stem cells co-localize. The complex cellular network within the necrotic region is believed to cause the resistance of the conventional chemotherapy and radiotherapy. Here we attempted to identify the role of WTAP in GBM and what kind of RNA modification is associated with the proliferation and stemness in GBM. Improvement of our understanding about post-transcriptional regulation in GBM will allow us to find out the key oncogenic switch in GBM, and development of therapeutics targeting this mechanism will provide better survival benefits.

Methods: 23 Patient-derived cell lines, two established GBM cell lines, and astrocyte was arrayed to find out the correlation between WTAP and cancer stem cells. shWTAP and WTAP overexpression vector were infected into patient-derived GBM cancer stem cell lines to test the effect of WTAP. Western blot and polymerase chain reaction analysis was performed to find out the change in genes which are related to cell proliferation and stemness. Limiting dilution assay was performed to validate the function of WTAP in sphere-forming ability. RNA sequencing was performed on shWTAP infected patient-derived cancer stem cell to identify the type of RNA modification made by knocking down WTAP.

Results: Knocking down of WTAP using shWTAP in patient-derived cancer stem cell lines, we were able to see the decrease in cell proliferation and stemness-related genes while overexpressing WTAP in these cells slightly increased cell proliferation and stemness. WTAP knockdown cells had a significant decrease in sphere-forming ability. RNA sequencing of shWTAP cells has revealed the increase in intron retention and the decrease in exon skipping, which shows that WTAP has a role in decreasing intron retention while increasing exon skipping.

Conclusion: We have revealed that WTAP has an important role in increasing proliferation and maintaining stemness in GBM. We have also identified that WTAP decreases intron retention and increasing exon skipping.

Citation Format: Sae Whan Park, Young Taek Oh, Jong Bae Park, Jong Heon Kim. Wilms' tumor 1-associating protein increases cancer cell proliferation and stem-cell like properties through RNA modification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4442.
Abstract 4450: Integrative genomic and transcriptomic analysis of CHD family chromatin remodelers in human cancers: Initial evidence of an oncogenic role for CHD7
Authors: Yang Zeng-Quan , Chu Xiaofang , Guo hui , Jiang Yuanyuan , Yu Huimei , Liu Lanxin , Shan Wenqi
Published: August 2018

Chromodomain helicase DNA binding proteins (CHDs) are characterized by N-terminal tandem chromodomains and a central adenosine triphosphate-dependent helicase domain. CHDs govern the cellular machinery's access to DNA, thereby playing critical roles in various cellular processes including transcription, proliferation, and DNA damage repair. Accumulating evidence demonstrates that mutation and dysregulation of CHDs are implicated in the pathogenesis of developmental disorders and cancer. However, we know little about genomic and transcriptomic alterations and the clinical significance of most CHDs in human cancer. We used TCGA and METABRIC datasets to perform integrated genomic and transcriptomic analyses of nine CHD genes in more than 10 000 primary cancer specimens from 32 tumor types, focusing on breast cancers. We identified associations among recurrent copy number alteration, gene expression, clinicopathological features, and patient survival. We found that CHD7 was the most commonly gained/amplified and mutated, whereas CHD3 was the most deleted across the majority of tumor types, including breast cancer. Overexpression of CHD7 was more prevalent in aggressive subtypes of breast cancer and was significantly correlated with high tumor grade and poor prognosis. CHD7 is required to maintain open, accessible chromatin, thus providing fine-tuning of transcriptional regulation of certain classes of genes. We found that CHD7 expression was positively correlated with a small subset of classical oncogenes, notably NRAS, in breast cancer. Knockdown of CHD7 inhibits cell proliferation and decreases gene expression of several CHD7 targets, including NRAS, in breast cancer cell lines. Thus, our results demonstrate the oncogenic potential of CHD7 and its association with poor prognostic parameters in human cancer.

Citation Format: Zeng-Quan Yang, Xiaofang Chu, hui Guo, Yuanyuan Jiang, Huimei Yu, Lanxin Liu, Wenqi Shan,. Integrative genomic and transcriptomic analysis of CHD family chromatin remodelers in human cancers: Initial evidence of an oncogenic role for CHD7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4450.
Abstract 4451: Dynamic translational regulation of cancer-associated genes through mRNA structure
Authors: Lackey Lela Lynn , Coria Aaztli , Laederach Alain
Published: August 2018

Our goal is to understand how disrupting translational regulation leads to cancer by analyzing the extent to which RNA structure contributes to the translational efficiency of mRNAs in different phases of the cell cycle. We hypothesize that RNA structure is a dynamic projection of the cellular environment and actively contributes to gene regulation by combining sequence-based and environment-induced structures to influence post-transcriptional regulation. We focus on cell cycle regulation as a common, dynamic cellular process that is highly relevant to cancer development and progression. Additionally, during the cell cycle the translational efficiency of many different mRNAs have been shown to change from one phase to the next. We have determined the secondary structures of eight translationally regulated 5'UTRs using SHAPE-MaP (Selective 2' Hydroxyl Acylation by Primer Extension and Mutational Profiling). This chemical structure probing technique is unique in that it reads through chemical modifications during reverse transcription and can be coupled to next generation sequencing and incorporated into RNA structural modeling. Furthermore, we are able to probe these structures in vivo during different stages of the cell cycle. Our data show important conformational rearrangements in 5'UTRs between G1-phase cells and S-phase cells. These results suggest that mRNA structure is a dynamic attribute that plays a role in translational regulation. Many genes involved in cell cycle regulation are implicated in cancer development and progression. Our results confirm that RNA structure of 5' UTRs is an important component of translation regulation and ultimately controls protein expression levels.

Citation Format: Lela Lynn Lackey, Aaztli Coria, Alain Laederach. Dynamic translational regulation of cancer-associated genes through mRNA structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4451.
Abstract 4452: Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC
Authors: Makhov Peter , Kudinov Alexander , Deneka Alexander , Egleston Brian L. , Nicolas Emmanuelle , Cai Kathy Q. , Brebion Rohan , Avril Eleanor , Hitrik Mark , Nikonova Anna S. , Serebriiskii Ilya G. , Khazak Vladimir , Borghaei Hossein , Golemis Erica A. , Boumber Yanis
Published: August 2018

Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant ~2.7-fold upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was approximately correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. NSCLC lung tissue microarray analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer.

Citation Format: Peter Makhov, Alexander Kudinov, Alexander Deneka, Brian L. Egleston, Emmanuelle Nicolas, Kathy Q. Cai, Rohan Brebion, Eleanor Avril, Mark Hitrik, Anna S. Nikonova, Ilya G. Serebriiskii, Vladimir Khazak, Hossein Borghaei, Erica A. Golemis, Yanis Boumber. Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4452.
Abstract 4454: Ecdysoneless, a novel regulator of cell cycle progression and oncogenesis
Authors: Saleem Irfana , Mirza Sameer , Sarkar Aniruddha , Band Hamid , Band Vimla
Published: August 2018

Precisely regulated cell proliferation is essential for embryonic development and for homeostasis in adult organs and tissues, whereas uncontrolled cell proliferation is a hallmark of cancer. We have identified the human orthologue of Drosophila Ecdysoneless (ECD) as a critical regulator of cell cycle progression and oncogenesis. ECD is overexpressed in cancers of several organs, and its overexpression correlates with shorter survival in breast cancer patients. Overexpression of ECD leads to an oncogenic phenotype in immortal mammary epithelial cells. ECD deletion in breast cancer cells on the other hand decreased the in vitro oncogenic phenotype and the in vivo tumorigenesis.

Mechanistically, we showed that ECD associates with a protein complex called R2TP, composed of RUVBL1, RUVBL2, RPAP3, PIHID1 proteins, which is involved in several biological functions, including the dynamic assembly of protein-protein and protein-RNA complexes. In addition, affinity purification followed by Mass Spec analyses showed that ECD binds to several proteins, including PRPF8, that are components of critical protein complexes required for RNA splicing. Based on these findings, we hypothesize that ECD interacts with the R2TP complex and protein complexes that control key steps in the cellular mRNA processing. We hypothesize that these complexes are required for the overexpressed ECD to regulate mRNA processing to promote cell proliferation and cell survival in cancer cells. We will present data to document the ECD complexes required for cell cycle regulation and cell survival. Our studies reveal novel roles of ECD in cancer cells, and support the rationale for use of overexpression of ECD and its interacting proteins as molecular biomarkers of tumor progression as well as explore the targeting of these complexes to develop novel cancer therapy agents.

Citation Format: Irfana Saleem, Sameer Mirza, Aniruddha Sarkar, Hamid Band, Vimla Band. Ecdysoneless, a novel regulator of cell cycle progression and oncogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4454.
Abstract 4471: Pax9 regulates squamous cell differentiation and alcohol-associated carcinogenesis in the oro-esophageal epithelium
Authors: Xiong Zhaohui , Ren Shuang , Chen Hao , Liu Yao , Huang Caizhi , Zhang Yawan Lyvia , Odera Joab Otieno , Chen Tong , Kist Ralf , Peters Heiko , Garman Katherine , Sun Zheng , Chen Xiaoxin Luke
Published: August 2018

Pax9 is a transcription factor of the Pax family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of Pax9 in squamous cell differentiation and carcinogenesis of oro-esophageal epithelium. However, its functional role in differentiation and carcinogenesis remains unclear. In this study, Pax9 deficiency in mouse esophagus promoted cell proliferation, delayed cell differentiation and altered the global gene expression profile. Ethanol exposure down-regulated Pax9 expression in human esophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was down-regulated in human oro-esophageal squamous cell carcinoma (OESCC), and its down-regulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with an isocaloric Lieber-DeCarli liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted NMBA-induced squamous cell carcinogenesis in mouse tongue, esophagus, and forestomach. In conclusion, Pax9 regulates squamous cell differentiation in the oro-esophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. Pax9 down-regulation, at least in part, contributes to alcohol-associated oro-esophageal squamous cell carcinogenesis.

Citation Format: Zhaohui Xiong, Shuang Ren, Hao Chen, Yao Liu, Caizhi Huang, Yawan Lyvia Zhang, Joab Otieno Odera, Tong Chen, Ralf Kist, Heiko Peters, Katherine Garman, Zheng Sun, Xiaoxin Luke Chen. Pax9 regulates squamous cell differentiation and alcohol-associated carcinogenesis in the oro-esophageal epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4471.
Abstract 4472: Downregulation of POTEG predicts poor prognosis in esophageal squamous cell carcinoma
Authors: Li Yan
Published: August 2018

POTE ankyrin domain family, member G (POTEG) belongs to POTE family. The POTE gene family encodes very closely related proteins that are highly expressed in prostate, ovary, testis, and placenta. Recent studies indicated that the POTE proteins have a pro-apoptotic function. To examine whether POTEG also underwent apoptosis in ESCC cancer cells and the functions of POTEG in the development of esophageal squamous cell carcinoma (ESCC), we determined the location and expression of POTEG in ESCC cancer cells. Clinical association studies determined that POTEG downregulation was associated with poor clinical outcomes. Ectopic re-expression of POTEG effectively suppressed the tumorigenesis and metastasis of ESCC cells in vitro and in vivo, including the inhibition of cell growth rate, foci formation, soft agar colony formation, migration, invasion and tumor formation in nude mice. Molecular analyses revealed that POTEG downregulated CDKs, leading to subsequent inhibition of Rb phosphorylation, and consequently arrested Cell Cycle at G1/S Checkpoint. Prospectively, POTEG can reduce the level of anti-apoptotic proteins, while promote the level of proapoptotic proteins and inducing apoptosis. On the other side, POTEG inhibit metastasis by reducing epithelial-mesenchymal transition in ESCCs. Taken together, our results revealed that POTEG has a pivotal function in ESCC pathogenesis, with possible use as a biomarker and intervention point for new therapeutic strategies.

Citation Format: Yan Li. Downregulation of POTEG predicts poor prognosis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4472.
Abstract 4474: An intricate role of p53 and p21 in cellular alterations and drug penetration in spheroids of colorectal cancer cells
Authors: Das Viswanath , Annadurai Narendran , Hajdúch Marián
Published: August 2018

The tumor protein p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 (p21) are two proteins with an intriguing and a paradoxical role in cancer. Through p53-dependent signals, p21 negatively controls cell cycle progression to protect cells against deleterious effects of DNA damage. Although p53 is a tumor suppressor protein, recent evidence shows that some patients with wild-type (wt) p53 develop aggressive and drug-resistant cancers. Similarly, recent studies also suggest the oncogenic role of p21. In a p53 deficient background, p21 results in the development of a subset of highly proliferative and resistant population of cancer cells.

In this study, we examined the role of p53 and p21 in inducing cellular alterations using a panel of colorectal carcinoma cell lines with different p53 and p21 background and spheroid and spheroid-derived cell cultures. First, our data show that despite the presence of proliferating cells, spheroid-derived cultures of HCT116/p53wt cells continue to display an elevated p21 level even after 6 days of culture as a monolayer. These p21-overexpressing proliferating cells show increased resistance to doxorubicin and paclitaxel in comparison to their parental monolayer counterpart. In contrast to HCT116/p53wt cells, DLD-1/p53mut cells did not show elevated p21 levels following reversal from spheroid to monolayer. The increased p21 level in spheroids and spheroid-derived cultures of HCT116/p53wt cells led us to examine the role of p53 and p21 in drug penetration. Spheroids of HCT116/p53wt cells were less permeable to doxorubicin and showed increased expression of intracellular junctional (IJ) proteins (adherens and tight junctions). Knocking-out p21 in HCT116/p53wt cells decreased the level of IJ proteins, resulting in an increased doxorubicin penetration. Re-expression of p21 in p21-knocked-out HCT116/p53wt cells restored IJ protein levels, reducing doxorubicin penetration and the subsequent effect of doxorubicin on spheroids. Interestingly, however, knocking-out p21 in DLD-1/p53mut cells neither altered intracellular junctions nor affected doxorubicin penetration. These data indicate that the effect of p21 on IJ proteins and drug penetration is pertinent only in cells with wt p53. Overall, the data from our study suggest an intricate interplay between wt p53 and p21 in inducing cellular alterations that affect the sensitivity of cells to anti-cancer drugs in spheroids.

Citation Format: Viswanath Das, Narendran Annadurai, Marián Hajdúch. An intricate role of p53 and p21 in cellular alterations and drug penetration in spheroids of colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4474.
Abstract 4475: Adenomatous polyposis coli like protein (APCLP) functions as a novel negative regulator of NF-kB signaling in colon cancer cells
Authors: Martin Matthew , Mundade Rasika , Lu Tao
Published: August 2018

Colon cancer (CRC) is the second leading cause of cancer related deaths in the United States. CRC is marked by aberrantly activated signaling of the nuclear factor of kappaB (NF-kB), a family of transcription factors which regulate wide varieties of cellular processes. Despite recent advances in comprehension of players of NF-kB signaling, deeper understanding of its regulation is imperative for the development of novel cancer therapeutics. Using the powerful validation-based insertional mutagenesis (VBIM) technique, we recently discovered APCLP as a novel negative regulator of NF-kB. The objective of this study is to elucidate the role APCLP in regulating NF-kB signaling in CRC at the molecular and biological levels and to understand the mechanism by which this regulation occurs. To determine the biological effect of APCLP on NF-kB signaling, we used lentiviral vectors to either overexpress or knockdown (shRNA) APCLP in human CRC cell lines (HT-29, HCT116, DLD-1). We show that overexpression of APCLP decreased the NF-kB activity, reduced cellular proliferation, migratory ability, as well as anchorage-independent growth of cells while knockdown of APCLP had an inverse effect. Furthermore, in vivo experiments in a xenograft mouse model confirmed that APCLP overexpression impeded whereas shRNA knockdown promoted tumor growth. To study the mechanism by which APCLP regulated NF-kB signaling, we conducted co-Immunoprecipitation experiments and confirmed that APCLP and the major subunit of NF-kB, p65, may complex or bind directly to each other. Studies are ongoing regarding the mechanism of interaction between APCLP and p65. In summary, discovery of APCLP and understanding of its molecular mechanism and biological function are significant because the knowledge acquired from this study could lead to utilization of APCLP as a potential biomarker and therapeutic target in CRC as well as other cancers that are driven by hyperactivated NF-kB.

Citation Format: Matthew Martin, Rasika Mundade, Tao Lu. Adenomatous polyposis coli like protein (APCLP) functions as a novel negative regulator of NF-kB signaling in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4475.
Abstract 4478: The tumor suppressive function of HPP1 is mediated by its EGF-like domain
Authors: Elahi Abul , Ajidahun Abidemi , Hendrick Leah , Husain S. Mazher , Getun Irina , Becker Andreas , Glazer Evan S. , Shibata David
Published: August 2018

Introduction:

HPP1 is a putative tumor suppressor gene that has had significant interest as a potential serum- and stool-based biomarker in colorectal cancer. It is an Epidermal Growth Factor (EGF)-like ligand that we have previously demonstrated to mediate its effects via an erbB4-JAK-STAT signaling cascade. HPP1 is a secreted transmembrane protein comprised of two follistatin modules, a cytosolic tail with a potential G - protein-activating motif and an EGF-like domain which differs from EGF by replacement of arginine (R) by a histidine (H) residue at the critical 299 position. The exact component of HPP1 responsible for its biologic function has yet to be elucidated. We sought to examine the specific role of the EGF-like domain and the contribution of the H299R alteration.

Methods:

Synthetic wild-type (WT; H299) and mutant (R299) peptides were prepared at our crystallographic core facility. The change in amino acid did not alter the predicted 3D structure of the peptide. A total of 50ng/ml of each peptide per well (6-well plate) were used to treat the HPP1 non-expressing HCT116 colon cancer cell line. Cells were starved for 24 hours with a peptide exposure time of 7 minutes. Using immunoblot analyses, we examined alterations in the expression and activation status of erbB4, and STAT family proteins.

Results:

Delivery of the WT peptide into the HCT116 cell line resulted in increased activation/phosphorylation of erbB4, STAT1 and STAT2 with a concomitant downregulation of STAT3 activation as compared to controls. These findings are identical to the signaling profile that we have previously described for full length HPP1 and required for its tumor suppressive activity. The mutant peptide did not result in any change in the phosphorylation status of erbB4, STAT1 or STAT2 but was associated with an increase in STAT3 activation as compared to controls.

Conclusion:

The EGF-like domain of HPP1 is the key moiety responsible for triggering its tumor suppressive erbB -STAT signaling cascade. Furthermore, the histidine residue at position 299 is critical and differentiates it from the oncogenic function of EGF. Our findings may have implications for the further understanding of EGF-related signaling in cancer progression.

Citation Format: Abul Elahi, Abidemi Ajidahun, Leah Hendrick, S. Mazher Husain, Irina Getun, Andreas Becker, Evan S. Glazer, David Shibata. The tumor suppressive function of HPP1 is mediated by its EGF-like domain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4478.
Abstract 4493: p190B RhoGAP modifies growth factor signaling in breast cancer in a non-cell autonomous manner
Authors: Castro Eduardo , Preston Aaron , Quinlan Mykal , McHenry Peter
Published: August 2018

Estrogen mediates the growth and differentiation of breast epithelial cells and plays an important role in breast cancer progression. The upregulation of estrogen receptors has been linked to p190B RhoGAP in vivo. p190B regulates the activity of several Rho GTPases in vitro. To better understand the molecular mechanism of p190B's role in tumor progression, we sought to develop a cell culture model that would recapitulate the effects of p190B on various growth factor signaling pathways. Protein expression of p190B was knocked down using siRNA in MCF-7 breast cancer cells cultured in the absence of steroid hormones. Upon stimulation with estrogen, cell proliferation did not significantly differ between MCF-7 cells expressing reduced p190B and those expressing normal levels of p190B, suggesting that the effects of p190B on estrogen receptor expression are not cell autonomous and that additional components of the tumor microenvironment are required to recapitulate the in vivo findings.

Citation Format: Eduardo Castro, Aaron Preston, Mykal Quinlan, Peter McHenry. p190B RhoGAP modifies growth factor signaling in breast cancer in a non-cell autonomous manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4493.
Abstract 4495: A CD10+GPR77+ subset of cancer-associated fibroblasts promotes cancer formation and chemoresistance by sustaining cancer stem cells
Authors: Song Erwei
Published: August 2018

Carcinoma-associated fibroblasts (CAFs) are an abundant and heterogeneous population of stromal cells in the tumor microenvironment critically involved in cancer progression. However, the lack of effective tools to isolate CAF subsets has prevented a comprehensive functional characterization of CAFs as well as their therapeutic potentials. Here, we demonstrate that two cell-surface molecules, CD10 and GPR77, specifically define a new CAF subset correlated with chemoresistance and poor survival in multiple cohorts of breast and lung cancer patients. CD10+GPR77+ CAFs promote tumor formation and chemoresistance by providing a survival niche for the cancer stem cells (CSCs). Mechanistically the functions of the CAF subset are driven by persistent NF-κB activation via p65 phosphorylation and acetylation, which is maintained by complement signaling via GPR77, a C5a receptor. Furthermore, CD10+GPR77+ CAFs are crucial for the successful engraftment of patient-derived xenografts (PDXs) in vivo and targeting these CAFs with a neutralizing anti-GPR77 antibody abolishes tumor formation and restores tumor chemosensitivity. This discovery represents the first time a functional CAF subset is identified using specific surface markers, which will greatly facilitate future investigations of the tumor microenvironment and suggests that targeting the CD10+GPR77+ CAF subset could be an effective therapeutic strategy against CSCs-driven solid tumors.

Citation Format: Erwei Song. A CD10+GPR77+ subset of cancer-associated fibroblasts promotes cancer formation and chemoresistance by sustaining cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4495.
Abstract 4504: Functional roles of KRT6-KRT14 fusion variant 7 in desmosome remodeling and cell spreading/migration
Authors: Sheu Jim J. , Kuo Brian Y. , Wu Chia-Cheng , Yang Jacky , Chen Chih-Mei
Published: August 2018

Keratins are intermediate filament (IF) proteins with major functions in controlling/maintaining cell morphology and stiffness. Recent studies indicated possible involvement of keratin IFs in cancer developments. In our previous study, we have identified the existence of keratin fusions between KRT6 and KRT14 genes in oral squamous cell carcinoma (OSCC), and patients with the history of chewing betel nut showed higher frequency to harbor such fusions in their tumor tissues. In this study, we studied bio-functional roles of a major keratin fusion isotype K6-K14/V7, which correlated with bigger tumor size, in cell migration and invasion. We established a tet-off inducible gene expression system in OSCC cells. After gene being turned on, K6-K14/V7 formed a peripheral-dominant network that subsequently up-regulated genes related to actin filament remodeling via mechano-transduction. Actin-associated protrusion at cell leading edge was also increased by K6-K14/V7 to enhance cell migration and invasion. Interestingly, the desmosome junction was found to be dramatically reduced, leading to loss of cell-cell contact and stemness formation via epithelial-mesenchymal transition (EMT). The K6-K14/V7-expressing cells showed reduced drug sensitivity toward 5-FU and cisplatin treatment, and formed more colonies in vitro and bigger tumors in vivo. This study uncovers new functions/definitions for keratin IF in cancer development, Mechanisms revealed by this study can help us to design new strategies for new drug development and clinical treatments.

Citation Format: Jim J. Sheu, Brian Y. Kuo, Chia-Cheng Wu, Jacky Yang, Chih-Mei Chen. Functional roles of KRT6-KRT14 fusion variant 7 in desmosome remodeling and cell spreading/migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4504.
Abstract 4507: C-X-C motif ligand 1 from melanoma cells downregulates the invasion of their metastatic melanoma cells
Authors: Hatano Takaharu , Yashiro Masakazu , Motomura Hisashi , Fujikawa Heishiro , Maeda Shusaku
Published: August 2018

[Introduction]The prognosis of patients with malignant melanoma is extremely poor because of the frequent metastasis. We have occasionally experienced the clinical cases that malignant melanoma patients rapidly developed distant metastases after the surgical resection of primary cancer lesion. These cases suggested that a primary tumor may control a metastatic tumor. We hypothesized that primary melanoma cells might produce a factor(s) which regulate the progression of metastatic melanoma cells. We reported that the conditioned medium from B16 cells significantly (p=0.02) suppressed the invasion ability of B16/BL6 cells, which is a subline of B16 melanoma and shows high metastatic potential to lung at the previous meeting. Then we investigated the growth and invasion interaction between primary malignant melanoma cells and high metastatic melanoma cells at the molecular level. [Materials and methods]We used two mouse melanoma cell lines, B16 and B16/BL6, that latter of which is a subline of B16 melanoma and shows high metastatic potential to lung. We investigated the interaction between the parent B16 cells and daughter B16/BL6 cells by invasion assay, cell morphology, cytokine array, RT-PCR, and gelatin-zymography. [Results]The conditioned medium (CM) from B16 cells significantly (p=0.02) inhibited the invasion ability of B16/BL6 cells. The morphology of the B16/BL6 cells was changed by the CM from bipolar in B16 cells to a multipolar shape. The B16 cells produced high levels of C-X-C motif ligand 1 (CXCL1), CXCL10, and M-CSF compared to the B16/BL6 cells. CXCL1 significantly (p=0.01) decreased the invasion ability of B16/BL6 cells, but CXCL10 and M-CSF did not. The invasion-inhibitory activity of the CM from B16 cells was significantly (p=0.046) suppressed following the addition of a neutralizing anti-CXCL1 antibody. The CM of B16 and CXCL1 increased the E-cadherin mRNA level and MMP2 activity of B16/BL6 cells. [Conclusion]Primary melanoma cells might down-regulate the invasion activity of metastatic melanoma cells through CXCL1 signaling.

Citation Format: Takaharu Hatano, Masakazu Yashiro, Hisashi Motomura, Heishiro Fujikawa, Shusaku Maeda. C-X-C motif ligand 1 from melanoma cells downregulates the invasion of their metastatic melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4507.
Abstract 4510: SHH and GATA interplay: A potential therapeutic target for prostate cancer
Authors: Morsi Rami Z. , Kheir Wassim Abou , El-Khatib Layal , Msheik Hiba , Daoud Georges
Published: August 2018

Background: Prostate cancer is the second leading cause of cancer deaths in males worldwide. Typically, prostate cancer cells depend on androgens, and many therapeutic strategies target receptors involved in androgen regulation. However, certain castration-resistant tumors of the prostate are resistant to androgen deprivation therapies. Moreover, advanced-stage prostate tumors can proliferate uncontrollably either due to androgens or due to alteration in other signaling pathways. For instance, the activity of the sonic hedgehog (SHH) pathway, which usually decreases during post-natal development, is increased during prostate cancer progression. Of interest, a recent study reported a novel role for GATA transcription factors as potential repressors of SHH signaling. Based on the above-mentioned findings, we hypothesize that the balance between GATA and SHH pathways is perturbed in prostate cancer leading to overactivation of SHH pathways and its downstream effector proteins. Methods: We screened human prostate cancer cell lines, DU145, 22Rv1, and PC3, as well as a human immortalized normal prostate epithelial cell line, RWPE-1, for baseline levels of expression of SHH downstream effector proteins via real-time PCR using specific primers. Subsequently, we treated the cell lines with bone morphogenetic protein 4 (BMP4)-conditioned media in order to induce the expression of GATA transcription factors, and then we measured the levels of expression for each of the SHH pathway effector proteins by real-time PCR. Results: We demonstrated that GATA can interfere with the expression of SHH and its effector proteins in the human prostate cancer cell lines. Upon treatment with BMP4, we noted that there was a decrease in expression of Gli1, Gli2, and Gli3, downstream effectors of SHH, suggesting an anti-proliferative response. Additionally, Patched1 expression was also attenuated after treatment with BMP4. Similarly, Sox9 expression was also abrogated. Conclusion: Our results demonstrate that GATA transcription factors can act as potential repressors of the SHH pathway in the context of prostate cancer in vitro by interfering with the expression of SHH downstream effector proteins.

Citation Format: Rami Z. Morsi, Wassim Abou Kheir, Layal El-Khatib, Hiba Msheik, Georges Daoud. SHH and GATA interplay: A potential therapeutic target for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4510.
Abstract 4511: The role of Wnt5a signaling pathway in epithelial ovarian cancer progression
Authors: Asem Marwa , Young Allison , Oyama Carlysa , Burkhalter Rebecca , Buechler Steven , Miller Daniel , Stack Sharon
Published: August 2018

Ovarian cancer (OvCa) is the most fatal gynecologic malignancy and the 5th leading cause of cancer death among U.S. women. The majority of women with OvCa (75%) have a very low survival rate (30%), as OvCa is usually diagnosed in late stages after development of intraperitoneal metastasis. Thus, it is indispensable to understand the molecular mechanisms that contribute to OvCa metastatic success, in order to design effective treatment strategies to improve the overall survival of women with OvCa. Wnt5a is a noncanonical Wnt ligand that binds to several cell membrane receptors and activates many downstream signaling pathways that are fundamental for normal developmental processes during embryogenesis. In the past decade, the aberrant activation or inhibition of Wnt5a signaling is emerging as an important event in cancer progression, exerting both oncogenic and tumor-suppressive effects. The role of Wnt5a in OvCa is controversial, as studies report conflicting data. In addition, mechanistic data regarding the contribution of Wnt5a to OvCa progression are largely unavailable. The main aim of this research is to obtain a molecular-level understanding of Wnt5a signaling in OvCa and to investigate its potential roles in influencing OvCa metastatic success. Our data show Wnt5a is prevalent in ascites samples from women in different stages with OvCa, suggesting a role for Wnt5a in promoting disease progression. Data obtained from TCGA (n=583) show high expression of Wnt5a in primary ovarian tumors. Furthermore, Wnt5a enhanced OvCa cells adhesion, migration and invasion in a panel of organotypic and ex vivo functional assays. This was combined with striking morphologic changes characteristic of an invasive phenotype in OvCa cells treated with recombinant Wnt5a protein and formation of tunneling nanotubes (TNT). Overall, our data suggest that Wnt5a plays an oncogenic role in epithelial ovarian cancer cells. More experiments exploring Wnt5a activated pathways and effects in epithelial ovarian cancer cells are under way.

Citation Format: Marwa Asem, Allison Young, Carlysa Oyama, Rebecca Burkhalter, Steven Buechler, Daniel Miller, Sharon Stack. The role of Wnt5a signaling pathway in epithelial ovarian cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4511.