bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3789: Simultaneous visual assessment of RNA and protein expression in circulating tumor cells using the AccuCyte-Cytefinder system
Authors: Ramirez Arturo B. , Crist Sarah B. , Yeats Tyler , Werbin Jeffrey L. , Stilwell Jackie L. , Kaldjian Eric P.
Published: July 2017

Background. Circulating tumor cells (CTCs) provide real-time information regarding patient tumor phenotype, including RNA expression. This information may be valuable in guiding care of cancer patients. Simultaneous RNA and protein assessment has been reported previously in large populations of cells. Here, we describe methods to simultaneously evaluate RNA and protein expression on rare single CTCs using the AccuCyte – CyteFinder system (RareCyte).

Methods. To establish RNA detection in model CTCs (mCTCs), we used SkBr3 cells spiked into blood and the isolated buffy coat was processed by AccuCyte onto microscope slides. RNAscope® in-situ hybridization (ISH, Advanced Cell Diagnostics) was performed to detect Her2, UBC (positive control) and dapB (negative control) expression in mCTCs. A process to simultaneously stain for RNA and protein was then developed to allow identification of mCTCs by protein expression of cytokeratin (CK) and EpCAM and measurement of gene expression with RNAscope. Various blood collection tubes were tested to measure gene expression, protein expression, and cell recovery up to 48 hours after blood draw. Image analysis software was developed to automatically analyze and count RNA dot number from 40x z-stack images of mCTCs. Finally, clinical samples were stained with the combined RNA/protein assay.

Results. Using RNAscope, Her2 and UBC expression could be detected in all SkBr3 cells and was negative in surrounding white blood cells (WBC). Using blood collected into EDTA tubes and processed immediately after spike-in, there were an average of 29 Her2 mRNA dots and 20 UBC dots/cell; negative control dapB gave 1 dot/cell. Using CK and EpCAM expression for mCTCs identification, recovery of mCTCs was over 95%. The RNA/protein assay was compared using blood collected and stored in EDTA, CellSave®, Cell-Free DNA®, Cell-Free RNA®, Cyto-Chex®, and RareCyte BCT tubes. At 48 hours, Cell-Free RNA tubes had the highest number of RNA dots/cell – about half as many dots/cell compared to fresh EDTA samples. Clinical samples were successfully stained with the RNA/protein assay.

Conclusions. We have developed a protocol that identifies rare mCTCs using protein staining, and measures RNA expression by RNAscope ISH. This assay may be a useful clinical tool for the real-time investigation of CTC gene expression in cancer patients.

Citation Format: Arturo B. Ramirez, Sarah B. Crist, Tyler Yeats, Jeffrey L. Werbin, Jackie L. Stilwell, Eric P. Kaldjian. Simultaneous visual assessment of RNA and protein expression in circulating tumor cells using the AccuCyte-Cytefinder system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2017-3789
Abstract 3809: SWI/SNF aberrations sensitize pancreatic cancer cells to DNA crosslinking agents
Authors: Shen Zhewei , Davidson Jean , Pollack Jonathan
Published: July 2017

While gemcitabine has been the mainstay therapy for advanced pancreatic cancer, newer combination regimens (e.g. FOLFIRINOX) have extended patient survival, though carry greater toxicity. Biomarkers are needed to better stratify patients for appropriate therapy. Previously, we reported that one-third of pancreatic cancers harbor deletions or deleterious mutations in key subunits of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. The SWI/SNF complex mobilizes nucleosomes on DNA, and plays a key role in modulating DNA transcription and repair. Thus, we hypothesized that pancreatic cancers with SWI/SNF aberrations might exhibit compromised DNA repair, and show increased sensitivity to DNA damaging agents. Here, we studied pancreatic cancer cell lines with deficient (or else exogenously reconstituted) SWI/SNF subunits, as well as normal pancreatic epithelial cells (HPDE) following SWI/SNF subunit knockdown. Cells were challenged with DNA damaging agents, including those used in current combination regimens, and then cell viability assayed. We found that pancreatic cells with SWI/SNF dysfunction showed markedly increased sensitivity to DNA damaging agents, and in particular DNA crosslinking agents (cisplatin and oxaliplatin). Assaying clearance of γH2AX foci confirmed that SWI/SNF dysfunction impaired DNA damage response/repair. Finally, by analyzing pancreatic cancer patient data (TCGA), we found that pancreatic cancers with SWI/SNF deficiency (subunit mutation and/or decreased expression) showed a strong trend towards extended survival with platinum containing chemo regimens. Thus, SWI/SNF dysfunction sensitizes pancreatic cancer cells to DNA crosslinking agents, and SWI/SNF mutation status may provide a useful biomarker to predict which patients are likely to benefit from platinum-containing chemotherapy regimens.

Citation Format: Zhewei Shen, Jean Davidson, Jonathan Pollack. SWI/SNF aberrations sensitize pancreatic cancer cells to DNA crosslinking agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3809. doi:10.1158/1538-7445.AM2017-3809
Abstract 381: Genomic alterations in mucins in cancer
Authors: King Ryan J. , Yu Fang , Singh Pankaj K.
Published: July 2017

Mucins promote the growth, aggressiveness, and invasiveness of various cancer types. Changes in the expression or localization of mucins can significantly alter patient survival. Due to their predominance in multiple cancers, biomarkers and therapeutic agents have been developed to improve patient care. However, an extensive investigation across multiple cancer subtypes examining the genomic status of the cohort of mucins has not been performed and could yield significant leads into new roles of mucins in different cancers. Utilizing The Cancer Genome Atlas, the cancers of 11 mucin expressing tissues were examined for the genomic alterations in mutations, mRNA expression levels, copy numbers, methylation status, and the impact on patient survival due to these genomic features. It was discovered that different mucins across multiple tissue origins and cancer subtypes might have a different rate, pattern, and survival impact. MUC1 was not mutated in the majority of cancers; however, we did observe a frequent T112P mutation in pancreatic tumors that was also present in other cancers. MUC4 was frequently mutated at H4205 and MUC4 mutations were seen to have survival differences. Multiple significant differences were discovered in mRNA expression, which varied distinctly between cohorts. One such pattern was observed with de novo expression of MUC21 in colorectal cancers, in which mRNA expression was suddenly induced in cancer and increased in a stage dependent manner. Copy number alterations were seen for only a few cancer subtypes and mucins, including MUC4 in lung squamous cell carcinomas. Global promoter demethylation was generally observed across the cancers. MUC15 in renal cancers was seen to be one of the most demethylated mucins, in which mRNA was seen to be substantially decreased or expunged. This extensive study investigated the status of multiple mucins in multiple cancer settings and highlights a few noticeable mucins that are suggested to be further studied for roles in biomarkers, survival, and etiology for cancer progression.

Citation Format: Ryan J. King, Fang Yu, Pankaj K. Singh. Genomic alterations in mucins in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 381. doi:10.1158/1538-7445.AM2017-381
Abstract 3815: The clinical impact of c-MET over-expression in advanced biliary tract cancers
Authors: Heo Mi Hwa , Kim Seung Tae , Park Young Suk , Park Joon Oh , Lee Hansang
Published: July 2017

Background: c-MET is a proto-oncogene that encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF). Activation of HGF-c-MET signaling involves cell invasiveness and evokes metastasis through direct involvement of tumor angiogenesis. However, the value of c-MET overexpression is still unknown in metastatic BTC.

Methods: We analyzed the incidence and clinicopathologic characteristics of c-MET overexpression in advanced BTC. Moreover, we investigated the value of c-MET overexpression in predicting response to gemicitabine plus cisplatin (GC), a first line standard regimen, and as a prognostic marker in metastatic BTC.

Results: The BTC subtype distribution (N=44) was as follows: intrahepatic cholangiocarcinoma (IHCC, n=7), extrahepatic cholangiocarcinoma (EHCC, n=25) and gallbladder cancer (GBC, n=12). Liver (52.3%) was the predominant metastatic site, followed by lymph nodes (36.4%) and bone (15.9%). Among the 44 patients analyzed for c-MET expression, 15 (34.1%) exhibited c-MET overexpression in tumor tissues. There was no significant difference in the prevalence of c-MET overexpression among primary sites in EHCC (7/25, 28.0%), IHCC (3/7, 42.9%), and GBC (5/12, 41.7%). There was also no significant correlation between specific clinicopathological variables and c-MET expression. Comparing the tumor-response to GC according to c-MET expression (overexpression vs. non-overexpression), there was no significant difference in either RR or DCR (p=0.394 and 1.000, respectively). PFS for all 44 patients was 9.00 months (95%CI, 7.47-10.53 months) and there was no significant difference for PFS between patients with c-MET overexpression and those without (p=0.917). The median OS was 14.4 months (95% CI, 12.8-19.2). There was a significant difference in OS between patients with c-MET overexpression compared to those without (13.7 vs. 14.4 months, respectively; p=0.708).

Conclusions: c-MET overexpression was detected in 34.1% of advanced BTC patients irrespective of tumor location. c-MET overexpression did not predict response to GC or survival. Further studies are needed to fully elucidate the value of c-MET overexpression as a novel biomarker in these patients.

Citation Format: Mi Hwa Heo, Seung Tae Kim, Young Suk Park, Joon Oh Park, Hansang Lee. The clinical impact of c-MET over-expression in advanced biliary tract cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3815. doi:10.1158/1538-7445.AM2017-3815
Abstract 3816: Mutations of the lim protein ajuba mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell cycle inhibitors
Authors: Zhang Ming , Ratnakar Singh , Peng Shaohua , Tuhina Mazumdar , Li Shen , Tong Pan , Pickering Curtis , Myers Jeffrey N. , Wang Jing , Johnson Faye M.
Published: July 2017

The genomic alterations identified in head and neck squamous cell carcinoma (HNSCC) tumors have not resulted in any changes in clinical care, making the development of biomarker-driven targeted therapy for HNSCC a major translational gap in knowledge. To fill this gap, we used 59 molecularly characterized HNSCC cell lines and found that mutations of AJUBA,SMAD4 and RAS predicted sensitivity and resistance to treatment with inhibitors of polo-like kinase 1 (PLK1), checkpoint kinases 1 and 2, and WEE1. Inhibition or knockdown of PLK1 led to cell-cycle arrest at the G2/M transition and apoptosis in sensitive cell lines and decreased tumor growth in an orthotopic AJUBA-mutant HNSCC mouse model. AJUBA protein expression was undetectable in most AJUBA-mutant HNSCC cell lines, and total PLK1 protein expression was increased in cell lines wild-type for AJUBA. Exogenous expression of wild-type AJUBA in an AJUBA-mutant cell line partially rescued the phenotype of PLK1 inhibitor-induced apoptosis and decreased PLK1 substrate inhibition, suggesting a threshold effect in which higher drug doses are required to affect PLK1 substrate inhibition. PLK1 inhibition was an effective therapy for HNSCC in vitro and in vivo. However, biomarkers to guide such therapy are lacking. We identified AJUBA, SMAD4 and RAS mutations as potential candidate biomarkers of response of HNSCC to treatment with these mitotic inhibitors.

Citation Format: Ming Zhang, Singh Ratnakar, Shaohua Peng, Mazumdar Tuhina, Shen Li, Pan Tong, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Mutations of the lim protein ajuba mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell cycle inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3816. doi:10.1158/1538-7445.AM2017-3816
Abstract 3837: Primary tumor cell lines derived from gastric PDX tumors displays differences in chemosensitivity
Authors: Ge Xiaomei , Qiang Fulin , Zhang Yixin , Liu Jibing , Yang Lei , Qu Yingying , Xie Fubo , Li Xueting , Gu Ying , Zhou He
Published: July 2017

Gastric cancer is a multifactorial and fatal disease with limited effective therapeutic options. Its overall mortality ranked third among cancer-related deaths worldwide. Patient-derived-xenograft (PDX) models have been widely used in the biomedical research. They recapitulate the genomic diversity/heterogeneity of patient tumors, maintain a lot of the characteristics of the original tumors and showed much better correlation with clinical outcome comparing to traditional cell line derived xenograft models. However, the relatively low throughput and time-consuming nature of PDX studies prevent them from being used in large scale screening or mechanistic studies. To facilitate drug development in gastric cancer, different cancer cell lines have been developed in our laboratory from gastric PDX tumors. We performed STR analyses to validate their identity and RNAseq data also showed high similarities to their parental PDX tumors. These primary gastric tumor cell lines displayed differences from each other in their chemo-sensitivities in vitro and in vivo. They also differ in their protein expression such as the HER-2 expression level. Pharmacology studies using different compounds showed similar drug responses of these cell lines in tissue culture, xenografts using these cell lines and the original PDX models. Our data demonstrated the high fidelity of these cell lines to their parental PDX tumors and serve as a bridge between the high-throughput drug screening and highly clinical relevant PDX models and will greatly facilitate the drug development in gastric cancer.

Citation Format: Xiaomei Ge, Fulin Qiang, Yixin Zhang, Jibing Liu, Lei Yang, Yingying Qu, Fubo Xie, Xueting Li, Ying Gu, He Zhou. Primary tumor cell lines derived from gastric PDX tumors displays differences in chemosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3837. doi:10.1158/1538-7445.AM2017-3837
Abstract 3839: MLN8237 treatment in an orthoxenograft rodent model for malignant peripheral nerve sheath tumors
Authors: Mrowczynski Oliver
Published: July 2017

Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise from peripheral nerves and often occur in the extremities. MPNSTs arise sporadically or in the neurofibromatosis type one (NF1). NF1 is an autosomal-dominant disorder characterized by neurofibromas, cutaneous hyperpigmentation, and intellectual deficits affecting 1 in 3000 births. The lifetime risk of a person with NF1 developing MPNSTs ranges between 8-13%. MPNSTs have increased expression of the oncogene Aurora kinase A. This leads to enhanced cell proliferation, making MPNSTs extremely aggressive, with high potential for metastasis, and devastating prognosis, with five year survival estimates ranging from a dismal 15-60%. MPNSTs are currently treated with surgical resection, sometimes requiring limb amputation, as well as chemoradiation, all of which demonstrate limited effectiveness, and highlight the necessity for novel therapies. To evaluate the effectiveness of new treatments, a translationally accurate, robust, and cost-effective model is crucial. A major goal of this study was to produce an orthotopic xenograft murine MPNST model for evaluation of novel therapies, which we created by intra-neural injection of luciferase transfected MPNST cells into the mice sciatic nerve. Our model circumvents drawbacks of previous in vivo models by producing a robust tumor in the physiologically relevant microenvironment of the nerve that allows for accurate tumor burden quantification through luciferin fluorescence. Another goal of this study was to demonstrate the effectiveness of MLN8237 for MPNST treatment. MLN8237 is an aurora kinase A inhibitor that we demonstrate to decrease MPNST burden and increase survival significantly. This study provides a robust and reproducible MPNST model, allowing for the advancement of MPNST therapy. This study also demonstrates the effectiveness of MLN8237 for MPNST treatment. Together, our data has major implications on the future of MPNST research by providing a robust murine model as well providing evidence that MLN8237 is an effective treatment for MPNSTs.

Note: This abstract was not presented at the meeting.

Citation Format: Oliver Mrowczynski. MLN8237 treatment in an orthoxenograft rodent model for malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3839. doi:10.1158/1538-7445.AM2017-3839
Abstract 3850: Establishment of patient derived model of high-risk endometrial cancer for pre-clinical combined carboplatin and decitabine therapy
Authors: Feng Weiwei , Zhu Menghan , Yu Yinhua , Jiang Wei
Published: July 2017

Objective: The high risk endometrial cancers (EC), including high grade EC, serous carcinoma (SC), clear cell carcinoma (CC) and carcinosarcoma, account for 50% of death. Therapies for them are limited and patient derived tumor xenograft (PDX) model become useful tools to predict response to treatment. Here we compared the two methods of establishment of PDX models and evaluated the efficacy of carboplatin and decitabine in two high grade EC PDX models.

Methods: Fresh tumor tissues collected from 11 primary high risk EC patients (6 high grade ECs, 4 SCs, 1 CC and 1 carcinosarcoma) were engrafted subcutaneously and in subrenal capsule in SCID mice. For those subcutaneous models, tumors were then orthotopic transplanted into uterine cavity. Histology, vimentin, cytokeratin, ER, PR, P53 expression were evaluated, together with mutation profiles by next generation sequencing for 500 cancer-panel genes. The efficacy of carboplatin and decitabine were evaluated in two high grade EC models.

Results: The total tumor take rate was 81.8% (9 /11), without regard to engraft methods. The tumor take rate was higher in subrenal capsule models than subcutaneous models (70% vs 54.5%, respectively). The time for tumor formation (from transplantation to 1 cm in diameter) varies greatly from 4 weeks to 5 months. We observed good similarity between primary tumors and corresponding different passage of xenografts. One high grade EC model was sensitive to carboplatin and decitabine treatment whereas another was drug resistant. Two models harbor different genetic mutation profiles.

Conclusion: The high tumor take rate ensures the establishment of the high risk EC biobank which provides a powerful resource for pre-clinical drug-sensitive tests as well as identification of biomarkers of response or resistance.

Note: This abstract was not presented at the meeting.

Citation Format: Weiwei Feng, Menghan Zhu, Yinhua Yu, Wei Jiang. Establishment of patient derived model of high-risk endometrial cancer for pre-clinical combined carboplatin and decitabine therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3850. doi:10.1158/1538-7445.AM2017-3850
Abstract 3862: In vivo chemopreventive of pseudosemiglabrin via inhibition of tumor angiogenesis and manipulation of hormones in estrogen-responsive breast cancer
Authors: Hassan Loiy E. Ahmed
Published: July 2017

Breast cancer is the most frequently diagnosed form of malignancy and the second leading cause of death in Western women. Mortality and most of the complications associated with breast cancer are due to metastasis developing in regional lymph nodes and in distant organs, including bone, lung, liver and brain. Chemopreventive is administration of chemical agents to prevent the initiational and promotional events associated with carcinogenesis. Some chemopreventive agents can prevent reaction or attacking molecules by different ways, these agents can suppress growth factors or signaling pathway. On this study we examined (-)-Pseudosemiglabrin (SSG) a natural compound extracted from Tephrosia apollinea on breast cancer cells and tumor vascularization in vitro and in vivo. Hormone sensitive breast cancer cell MCF-7 was found subtitle susceptible to SSG, it showed profound antitumorgeneis via inhibiting breast cancer cell migration, invasion and colony formation in vitro settings. In silico investigation of potential PARP inhibitor, SSG showed high binding affinity comparable to Rucaparib (standard PARP inhibitor). The natural compound significantly inhibited the sprouting of new blood vessels in ex vivo rat aorta ring assay; the antiangiogenic efficacy is perturbed by targeting VEGF expression and VEGFR phosphorylation. SSG completely inhibited the onset of breast cancer in animals pretreated with the compound. Molecular docking study showed that SSG binds to active site of Aromatase (the enzyme responsible for a key step in the biosynthesis of estrogens) with binding affinity better than standard aromatase inhibitor (Arimidex), moreover the compound has inhibitory activity against estrogen receptor in the breast cancer cells comparable to Tamoxifen. Our findings reveal that (-)-Pseudosemiglabrin can potentially be beneficial in preventing breast cancer in high-risk women or it can be adjuvant therapy with standard chemotherapy or even can be used for the treatment of women who have been diagnosed with breast cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Loiy E. Ahmed Hassan. In vivo chemopreventive of pseudosemiglabrin via inhibition of tumor angiogenesis and manipulation of hormones in estrogen-responsive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3862. doi:10.1158/1538-7445.AM2017-3862
Abstract 3866: Investigating the role of class I HDACs in Ewing sarcoma pathology
Authors: Heyking Kristina von , Hensel Tim , Singer Julia , Schmidt Oxana , Burdach Stefan , Richter Günther H.
Published: July 2017

Introduction: Recent evidence demonstrated that a low mutation rate seems a general feature of pediatric cancers. Ewing sarcoma (ES) is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Other contributing somatic mutations involved in disease development have only been observed at low frequency. Thus, cancer in children is not solely a genetic disease and can neither be understood nor cured presumably without epigenetics. Previously, we identified the histone methyl-transferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the enzymatic subunit of the polycomb PRC2 complex, to be over-expressed in ES. RNA interference of EZH2 suppressed tumor development and metastasis in vivo and microarray analysis of EZH2 knock down revealed an EZH2-maintained, undifferentiated, reversible phenotype in ES.

Experimental procedures: The role of class I histone deacetylases (HDACs) was determined using different potent inhibitors like Trichostatin A (TSA), Romidepsin (FK22), Entinostat (MS-275) and PCI-34051 as well as a CRISPR/Cas9 approach to knock out specific HDACs. To analyze resulting changes qRT-PCR, Western Blotting, proliferation, apoptosis and invasion assays as well as immunofluorescence experiments were deployed.

Results: Interestingly, the effects of gene silencing after RNA interference of EZH2 could be mimicked by treatment of ES with TSA or MS-275. Microarray analysis revealed that treatment with MS-275 or TSA resulted in the induction of a similar pattern of differentiation genes as was previously observed after EZH2 blockade and seems to be dependent on histone deacetylase (HDAC) activity. Furthermore, in proliferation assays ES were more susceptible to treatment with HDAC inhibitors than other small round blue cell tumors such as neuroblastoma or pediatric cALL cells. Treatment of enzymatic inhibitors blocking EZH2 activity could not mimic the results observed after EZH2 RNA interference indicating that EZH2-containing PRC2 complexes may serve as a building block of class I HDAC activity in ES.

Conclusion: Class I HDACs seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in ES and thus interesting new treatment opportunities for this malignant disease.

Citation Format: Kristina von Heyking, Tim Hensel, Julia Singer, Oxana Schmidt, Stefan Burdach, Günther H. Richter. Investigating the role of class I HDACs in Ewing sarcoma pathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3866. doi:10.1158/1538-7445.AM2017-3866
Abstract 3868: The stem cell factor LIN28B regulates proliferation and apoptosis in diffuse intrinsic pontine glioma
Authors: Guo Huizi , Akhtarkhavari Sepehr , Eberhart Charles G. , Kaur Harpreet , Raabe Eric H.
Published: July 2017

Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable, invasive and aggressive pediatric brain tumor. Identifying molecular markers that regulate tumor growth and invasion are needed for developing efficient treatment strategies. LIN28B is a stem cell factor expressed during normal fetal development and re-expressed in cancer cells. We had previously shown that LIN28A, another family member of LIN28 proteins, regulates invasion and tumorigenicity in adult high grade gliomas. We observed increased LIN28B expression in patient-derived DIPG neurosphere cell lines using western blotting. We hypothesized that LIN28B promotes proliferation and prevents apoptosis in DIPG. Using two different lentiviral transduced short hairpin RNAs (shRNA), we suppressed LIN28B protein levels in DIPG neurospheres, as confirmed by western blotting. DIPG neurospheres that have been treated with LIN28B shRNA showed reduced proliferation as measured by BrdU incorporation (P<0.01) and increased apoptosis as measured by cleaved caspase-3 (CC-3) expression (P<0.01). To determine the molecular mechanism of LIN28B-mediated phenotypes in DIPG, we studied the canonical downstream effector of LIN28B called HMGA2, which is a DNA-binding protein that functions as a transcriptional regulator. Using western blotting, we found decreased HMGA2 protein levels in DIPG neurospheres infected with LIN28B shRNA. Taken together, our results suggest that LIN28B is important for promoting DIPG cell proliferation and preventing apoptotic cell death. Additionally, we also found that LIN28B regulates HMGA2 expression in DIPG neurospheres. Future studies will focus on expanding our understanding of the molecular mechanisms of LIN28B-regulated malignancy in DIPG.

Citation Format: Huizi Guo, Sepehr Akhtarkhavari, Charles G. Eberhart, Harpreet Kaur, Eric H. Raabe. The stem cell factor LIN28B regulates proliferation and apoptosis in diffuse intrinsic pontine glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3868. doi:10.1158/1538-7445.AM2017-3868
Abstract 3879: Expression of sox2 and oct4 in human oral squamous cell carcinoma and its relationship with clinical factors
Authors: Tamatani Tetsuya , Takamaru Natsumi , Ohe Go , Kudo Keiko , Kudo Takaharu , Takahashi Akira , Yamamura Yoshiko , Fujisawa Kenji , Nagai Hirokazu , Miyamoto Youji
Published: July 2017

Background: A cancer stem cell (cancer initiating cell, CSC) is considered capable of self -replication, self-differentiation, drug resistance, and immune evasion. Recently, CSC has become increasingly important in the treatment of malignant tumors. Cancer stem cells express specific molecules termed CSC marker, including sex determining region Y-box2 (SOX2), and octamer-binding transcription factor 4 (Oct4), and their expression has been reported to be the potential prognostic values. However, the prognostic values of SOX2 and Oct4 expression in patients with oral cancer are less understood. [Purpose]The aims of present study were to evaluate the expression of SOX2 and Oct4 in oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the CSC marker expression, clinical stages, histological differentiation, the classification of invasion mode, cerebral lymph node metastasis, distant metastasis, and disease-free survival rate.

Materials and Methods: Tissue specimens were obtains from 108 patients with OSCC after surgery or biopsy. Immunohistochemistry was used to assess SOX2 and Oct4 protein using at least 10% staining-positive cells as the definition of positive staining. Results: Immunohistochemical analysis of 108 cases showed that 42 cases (39%) expressed SOX2. There was no significant association between SOX2 expression and tumor size, invasion mode or histological differentiation. However, there was significant association between SOX2 expression and distant metastasis or disease-free survival rate at stage 1 and 2 patients (73 cases). Otherwise, seventy cases (65%) cases of 108 OSCC patients expressed Oct4. There was significant association between Oct4 expression and histological differentiation. There was no significant association between Oct4 expression and tumor size, invasion mode, metastasis, or disease-free survival rate.

Conclusions: These findings suggested that the expression of SOX2 may be good marker indicating survival in patients with OSCC.

Citation Format: Tetsuya Tamatani, Natsumi Takamaru, Go Ohe, Keiko Kudo, Takaharu Kudo, Akira Takahashi, Yoshiko Yamamura, Kenji Fujisawa, Hirokazu Nagai, Youji Miyamoto. Expression of sox2 and oct4 in human oral squamous cell carcinoma and its relationship with clinical factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3879. doi:10.1158/1538-7445.AM2017-3879
Abstract 3902: Interleukin-6 blockade, a novel cancer stem cell targeted therapy, attenuates lung cancer tissue construction
Authors: Ogawa Hiroyuki , Koyanagi-Aoi Michiyo , Maniwa Yoshimasa , Aoi Takashi
Published: July 2017

Background: Lung cancer stem cells are considered to be responsible for lung cancer progression and metastasis. However, little is known about how they actually promote aggressive behaviors of lung cancer. Methods: We transduced OCT3/4, SOX2, and KLF4 (hereafter, OSK) into a KRAS-mutated (G12S) human lung adenocarcinoma cell line (A549) using retrovirus vectors. Dome-shaped colonies appeared in the OSK transduced A549 cells from 10 to 15 days after transduction. The colonies were picked up and sub-cultured. We named them OSK-A549-Colony cells. We evaluated cancer stem cell properties in the OSK-A549-Colony cells in terms of their chemo resistance, and sphere formation ability. We also assessed organoids constructing ability by co-culture with mesenchymal stem cells (MSCs) and human umbilical vein epithelial cells (HUVECs) on low attachment plates. To clarify the molecular mechanisms that determined the properties of the OSK-A549-Colony cells, we performed microarray analysis. We further analyzed the molecular mechanisms of organoids construction by OSK-A549-Colony cells and searched for a method to destroy them. Results: The induced OSK-A549-colony cells were significantly more resistant to cisplatin than the parental A549 cells. Sphere forming ability was enhanced in the OSK-A549-Colony cells. Co-culture with MSCs and HUVECs showed that only the OSK-A549-Colony cells were able to construct consolidated organoids in vitro that mimicked bona-fide human lung cancer tissues. We named them “lung cancer organoids”. We found that interleukin-6 (IL-6), which was the most differentially expressed gene in the OSK-A549-Colony cells by microarray analysis, facilitated the formation of lung cancer organoids via the conversion of MSCs into alpha-smooth muscle actin (αSMA)-positive cells. Surprisingly, the combination of anti-IL-6 antibody and cisplatin could destroy the lung cancer organoids, while cisplatin alone could not. Conclusion: By transducing defined factors, A549 cells acquired lung cancer stem cell like properties. Analysis of chemosensitivity using the lung cancer organoids showed that the combination of IL-6 blockade and cisplatin could be a novel therapy targeting lung cancer stem cells.

Citation Format: Hiroyuki Ogawa, Michiyo Koyanagi-Aoi, Yoshimasa Maniwa, Takashi Aoi. Interleukin-6 blockade, a novel cancer stem cell targeted therapy, attenuates lung cancer tissue construction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3902. doi:10.1158/1538-7445.AM2017-3902
Abstract 3917: GenoCTC, a liquid biopsy device using microfluidic enrichment for affinity-based isolation of circulating tumor cells
Authors: Ryu Jiyeon , Kim Do Hyung , Kim Gyeong-Mi , Kim Jin Soo , Shin Youngkee , Lee Jaeku , Lee Hun Seok
Published: July 2017

To achieve a complete remission of cancer is still challenging that most patients with solid cancer ultimately experience metastasis. Detection of circulating tumor cells (CTCs) from the peripheral blood is promising key to solve the cancer cell dissemination and make accurate diagnosis, therefore, many efforts have been made to isolate the CTCs. Here, we newly developed the liquid biopsy device, GenoCTC, with microfluidic chips isolating CTCs from the patient whole blood. To isolate CTCs, EpCAM+ CTCs in the whole blood are first bound with magnetic beads conjugated with human-EpCAM antibody, and GenoCTC and its chip can isolate the EpCAM+ CTCs through magnetic based method. With 7.5 mL whole blood from lung cancer patient, we isolated EpCAM+ CTCs, and these CTCs were validated by immunofluorescence assay using anti-CK and anti-CD45. We also enumerated EpCAM+ CTCs in 17 whole blood samples from 10 lung cancer patients that various number, 2 to 114, of CTCs were found from each patient. The number of CTCs is known to have a correlation with disease progression and chemo-resistance, so the clinical meaning of this result is needed to be investigated. Furthermore, some CTCs are also found to be EpCAM independent which is regarded to have clinical utility involved in EMT. We used plastin-3 as its marker, and it was confirmed with HS 578T breast cancer cell line which shows low expression of EpCAM but high expression of plastin-3. The various molecules expressed on CTCs can be isolated with GenoCTC for further study. The characterization of CTCs is not only limited to EpCAM expression that it is important to be elucidated the complex biology of CTCs to apply companion diagnosis for personalized medicine.

Citation Format: Jiyeon Ryu, Do Hyung Kim, Gyeong-Mi Kim, Jin Soo Kim, Youngkee Shin, Jaeku Lee, Hun Seok Lee. GenoCTC, a liquid biopsy device using microfluidic enrichment for affinity-based isolation of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3917. doi:10.1158/1538-7445.AM2017-3917
Abstract 3919: Isolation and characterization of viable pancreatic cancer circulating tumor cells using size-based filtration system
Authors: Yu Kenneth H. , Krempley Benjamin D. , McCarthy Brian
Published: July 2017

Introduction: Circulating tumor cells (CTCs) are important actors in metastasis, a key feature of pancreatic ductal adenocarcinoma (PDAC). The ability to isolate, characterize and culture viable CTCs holds promise for understanding biology, treatment response and resistance.

Methods: Peripheral blood samples from healthy donors and patients with advanced PDAC were processed using an innovative size filtration system (Viatar LLC, Lowell MA). Normal human blood samples were spiked with varying numbers of human PDAC organoid cells. The impact of buffy coat separation was studied. Isolated CTCs were cultured in Matrigel to generate organoids as previously described. Cells were imaged with fluorescently labeled antibody to EpCAM.

Data: Using spiked normal human blood samples, viable CTCs can be isolated and expanded reliably in 3D organoid culture from as few as 10 CTCs/mL. Separation and processing of the buffy coat enriched for the number of viable CTCs isolated. EpCAM positive CTCs were isolated from 6/6 blood samples from patients with advanced PDAC. Updated results will be presented.

Conclusions: The current size-based filtration system allows for collection of viable CTCs from human PDAC blood samples. Current work will use this system to grow, expand and characterize human PDAC CTCs in organoid culture. Genetic, transcriptional, and epigenetic characteristics will be compared with the primary tumor. CTC characteristics will be studied as biomarker of treatment response and resistance.

Citation Format: Kenneth H. Yu, Benjamin D. Krempley, Brian McCarthy. Isolation and characterization of viable pancreatic cancer circulating tumor cells using size-based filtration system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3919. doi:10.1158/1538-7445.AM2017-3919
Abstract 3923: Oncogenic kinase-induced PKM2 Y105 phosphorylation promotes tumorigenesis and cancer stem cell-like property
Authors: Zhou Zhifen , Li Min , Zhang Lin , Yu Dihua
Published: July 2017

The role of pyruvate kinase M2 isoform (PKM2) in tumor progression is controversial. Previously, it was reported that PKM2 has an oncogenic function in promoting tumor growth in xenograft models; however, PKM2 was indicated non-oncogenic or even anti-oncogenic because depletion of PKM2 accelerates mammary tumor growth in the Brca1 knockout mouse model of breast cancer. Since tumors frequently harbor various abnormally activated oncogenic kinases, we hypothesized that phosphorylation of PKM2 by oncogenic kinases in tumor cells enables PKM2 to promote tumor growth, while unphosphorylated PKM2 in non-transformed cells cannot promote tumor growth. Here, we report that PKM2 proteins were largely unphosphorylated and formed tetramers in normal mammary epithelial cells (MCF10A, MCF12A). On the other hand, PKM2 proteins were phosphorylated at Y105 and formed dimers in breast cancer cells (MDA-MB-231, MDA-MB-435). Knocking down PKM2 didn’t affect normal mammary epithelial cell (MCF10A) growth but significantly decreased breast cancer cell MDA-MB-231 proliferation. In addition, introducing the PKM2-Y105D phosphomimetic mutant into MCF10A cells induced epithelial to mesenchymal transition, increased CD44Hi/CD24Neg stem cell-like cell population, and induced colony formation in soft agar, promoted cell migration and invasion. Interestingly, PKM2-Y105D increased YAP1 protein nuclear localization in MCF10A cells, whereas inhibiting YAP1 by siRNA or a chemical inhibitor (Verteporfin) decreased PKM2-Y105D-induced cancer stem cell-like population and colony formation ability. The data indicate that YAP1 is a novel downstream target of pY105-PKM2 in promoting the stem-like property and triggering mammary tumor initiation. Next, we performed receptor tyrosine kinase array and identified several oncogenic tyrosine kinases (AXL, EPHA2, FAK, Src, JAK3 and so on) that effectively phosphorylates PKM2 at Y105. Currently, we are testing the efficacy of targeting these oncogenic tyrosine kinases in reversing the phosphorylation of PKM2 and blocking its tumor-promoting function. Together, our data indicate that oncogenic kinases-induced phospho-Y105-PKM2, but not the unphosphorylated PKM2, promotes mammary tumor initiation by increasing cancer stem cell populations.

Citation Format: Zhifen Zhou, Min Li, Lin Zhang, Dihua Yu. Oncogenic kinase-induced PKM2 Y105 phosphorylation promotes tumorigenesis and cancer stem cell-like property [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3923. doi:10.1158/1538-7445.AM2017-3923
Abstract 3933: Characterization of oncogenic activity of interferon-induced protein with tetratricopeptide repeats 1 and 3 in human oral squamous cell carcinoma progression
Authors: Pidugu Vijaya Kumar , Yen Ai-Hsin , Chen Ying-Chen , Wu Mei-Maan , Liu Chung-Ji , Lee Te-Chang
Published: July 2017

Interferon-induced protein with tetratricopeptide repeats (IFITs) family has multiple tetratricopeptide repeats with helix-turn-helix structural motifs that mediate a variety of protein-protein interactions. There are four IFIT genes that have been identified namely, ISG56/IFIT1, ISG54/IFIT2, ISG60/IFIT3, and ISG58/IFIT5. IFIT proteins have been documented to involve in a variety of cellular functions, such as cell proliferation, migration, virus-induced translation initiation, replication and double-stranded RNA signaling. Our previous study has demonstrated that IFIT2 inhibits oral squamous cell carcinoma (OSCC) metastasis. However, little is known about IFIT1 and IFIT3 in the progression of OSCC.

In the present study, we are investigating the functional role of IFIT1 and IFIT3 genes in OSCC tumor progression. Immunohistochemical analysis of 147 patients derived tumor specimens with OSCC revealed a strong correlation between IFIT1 and IFIT3 proteins (p<0.001). Clinicoplathological characteristics correlation of IFIT1 and IFIT3 expression levels were positively associated with regional lymph node invasion and nerve invasion. IFIT1 was also associated with venous invasion. Ectopic expression of IFIT1 and IFIT3 proteins in Ca9-22 and SAS cells inducing PKC, EGFR, and AKT activation, resulting in OSCC cells resistance to cisplatin but highly susceptible to tyrosine kinase inhibitor gefitinib, whereas IFIT1 and IFIT3 knockdown SCC25 cells have shown opposite effect. Interestingly, IFIT1 and IFIT3 expression is associated with enhanced cell invasion activity and altered expression of epithelial-mesenchymal transition markers. These data suggested that IFIT1 and IFIT3 may serve as potential biomarkers for the OSCC treatment. The study on how IFIT1 and IFIT3 cause PKC, EGFR, and AKT activation and resistance to DNA damaging agents is ongoing. Our further investigations may explain the molecular signaling mechanism of IFIT1 and IFIT3 in OSCC tumor progression.

Citation Format: Vijaya Kumar Pidugu, Ai-Hsin Yen, Ying-Chen Chen, Mei-Maan Wu, Chung-Ji Liu, Te-Chang Lee. Characterization of oncogenic activity of interferon-induced protein with tetratricopeptide repeats 1 and 3 in human oral squamous cell carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3933. doi:10.1158/1538-7445.AM2017-3933
Abstract 3937: Diversity index as a novel predictor of tumor progression in breast cancer
Authors: Chung Yul Ri , Kim Hyun Jeong , Kim Young A , Chang Mee Soo , Hwang Ki-Tae , Park So Yeon
Published: July 2017

Intratumoral genetic heterogeneity leads to tumor progression and therapeutic resistance. However, due to the difficulty associated with its assessment, application of intratumoral genetic heterogeneity as a prognostic or predictive marker is limited. We investigated the significance of Shannon diversity index as a tool for measuring genetic heterogeneity by performing fluorescence in situ hybridization of c-Myc gene in invasive breast cancers, and correlated the Shannon diversity index for c-Myc copy number variation with clinicopathologic features of tumor including patient survival. Shannon index for c-Myc copy number variation strongly correlated with average c-Myc copy number and was significantly higher in tumors with c-Myc genetic or regional heterogeneity than in those without c-Myc amplification. High Shannon index was associated with high histologic grade, lymphovascular invasion, p53 overexpression, high Ki-67 proliferation index, hormone receptor negativity, and HER2 amplification. In survival analyses, c-Myc amplification or c-Myc copy number gain was not associated with patient survival. However, a high level of Shannon index was associated with poor disease-free survival. In subgroup analyses, it was found to be an adverse prognostic factor in hormone receptor-positive group, but not in hormone receptor-negative group. In a validation set, high Shannon diversity index for c-Myc copy number variation was also found to be associated with poor survival of the patients. We further investigated the correlation with clinicopathologic features and predictive power of Shannon index using another gene, fibroblast growth factor receptor 1 (FGFR1) and observed that high Shannon index for FGFR1 gene copy number variation was an independent prognostic factor for poor clinical outcome in a whole group and in hormone receptor-positive subgroup. In conclusion, this study demonstrated that higher diversity index was associated with adverse pathologic parameters of breast cancer and poor clinical outcome suggesting that Shannon diversity index, which represents intratumoral genetic heterogeneity, can be used as a potential biomarker for tumor progression and prognostication in patients with breast cancer.

Citation Format: Yul Ri Chung, Hyun Jeong Kim, Young A Kim, Mee Soo Chang, Ki-Tae Hwang, So Yeon Park. Diversity index as a novel predictor of tumor progression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3937. doi:10.1158/1538-7445.AM2017-3937