bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2751: Identification of predisposition genes involved in thyroid and breast carcinomas in patients with family history of these tumors by whole exome sequencing
Authors: Pinheiro Maisa , Marchi Fabio , Drigo Sandra , Reis Patricia , Coutinho Luiz Lehmann , Andrade Sonia , Kowaslki Luis Paulo , Achatz Maria Isabel , Rogatto Silvia
Published: August 2015

A significant number of patients with family history of breast (BC) and papillary thyroid carcinomas (PTC) have been detected at Oncogenetics Dept of AC Camargo Cancer Center, SP, Brazil. The aim of this study was to identify genetic alterations in patients with family cancer history of BC and TC by using whole exome sequencing (WES) analysis. All patients included in this study fulfilled the inclusion criteria as follows: proband with non-medullary TC and/or BC with family cancer history of these tumors AND at least two 1st- or 2nd- degree relatives with one of these tumors either diagnosed <45 years old OR one 1st- relative with one of these tumors and proband diagnosed <40 years old AND history of other tumors. Exclusion criteria comprised female patients with BC treated by radiotherapy before TC diagnosis and probands affected with common mutations in major hereditary predisposition genes. Twenty-four blood samples and four tumor samples were included in WES analysis. Libraries were prepared by using Nextera Exome Enrichment kit following manufacturer's instructions and were sequenced in HiSeq2000 sequencer (Illumina). An average depth of 95x was obtained. The analysis focusing in single nucleotide polymophisms (SNPs) in high penetrance genes detected 12 SNPs in six genes. Particularly, two potential pathogenic SNPs mapped at BRCA1 gene, previously associated with PTC and BC, were detected in at least 25% of samples. The evaluation of rare pathogenic alterations shared by all cases revealed two single nucleotide variations (SNVs) mapped at HYDIN gene. Alternative variants of HYDIN gene were previously detected in serum of patients diagnosed with colorectal, lung, breast and thyroid tumors [Laske K et al, Cancer Immunol Res 1: 190, 2013]. Further analyses are needed to confirm these alterations as potential predisposition candidates associated with breast and thyroid hereditary carcinomas. Furthermore, the data generated in this study could be incorporated into the general knowledge of cancer genetics, allowing the development of prevention strategies, screening and genetic counseling in these families.

Financial Support: FAPESP (2013/01867-8 and 2014/03983-8) and CNPq (481132/2012-0)

Citation Format: Maisa Pinheiro, Fabio Marchi, Sandra Drigo, Patricia Reis, Luiz Lehmann Coutinho, Sonia Andrade, Luis Paulo Kowaslki, Maria Isabel Achatz, Silvia Rogatto. Identification of predisposition genes involved in thyroid and breast carcinomas in patients with family history of these tumors by whole exome sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2751. doi:10.1158/1538-7445.AM2015-2751
Abstract 2758: Cancer risk in relatives of nasopharyngeal carcinoma - A register-based cohort study in Sweden
Authors: Liu Zhiwei , Fang Fang , Chang Ellen T. , Ye Weimin
Published: August 2015

Background: Little is known about the cancer susceptibility among relatives of patients with nasopharyngeal carcinoma (NPC) in non-endemic areas. We conducted a register-based cohort study to assess the relative risks (RRs) of cancer in families (including both first- and second-degree relatives) of NPC patients, compared with other individuals of the general population in Sweden, a low-incidence area for NPC.

Method: The present study included 11,602,616 individuals that were identified from the Swedish Censuses in 1960, 1970, 1980, and 1990, and were born in Sweden (defined as “general population”). Cross-linking this population to the Swedish Cancer Register and the Swedish Multi-Generation Register, we identified 9,157 relatives (3,645 first-degree and 5,512 second-degree) of 1,211 NPC probands that were diagnosed between 1958 and 2009 and were the first NPC cases in their families. The occurrence of incident cancers in these relatives and other individuals of the general population during 1961-2009 was ascertained through the Swedish Cancer Register. RRs of cancer in the relatives of NPC probands, compared with other individuals from the general population were calculated from Poisson regression models.

Results: The RR of all cancers combined among the first- or second-degree relatives of NPC probands was 1.03 (95% confidence interval [CI] = 0.95 to 1.11, P = 0.50), compared with other individuals of the general population. First-degree relatives of NPC probands had higher risks of NPC (N = 2, RR = 4.29, 95% CI = 1.07 to 17.17), and cancers of the larynx (N = 5, RR = 2.53, 95% CI = 1.05 to 6.09), prostate (N = 76, RR = 1.35, 95% CI = 1.07 to 1.68), and thyroid (N = 10, RR = 2.44, 95% CI = 1.31 to 4.53). In addition, a higher risk of cancer of the salivary glands was observed among the first-degree relatives of NPC probands with undifferentiated carcinoma (N = 2, RR = 6.64, 95% CI = 1.66 to 26.57). In contrast, a decreased risk of colorectal cancer incidence among relatives of NPC probands has been observed (N = 43, RR = 0.71, 95% CI = 0.53 to 0.96).

Conclusion: The increased risk of NPC and some other cancers among the first-degree relatives of NPC patients may potentially be explained by shared genetic and environmental risk factors, the latter including Epstein-Barr virus infection and tobacco smoking. However, interpretation of these findings should be cautious given the small number of cancer cases observed among the relatives of NPC patients.

Citation Format: Zhiwei Liu, Fang Fang, Ellen T. Chang, Weimin Ye. Cancer risk in relatives of nasopharyngeal carcinoma - A register-based cohort study in Sweden. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2758. doi:10.1158/1538-7445.AM2015-2758
Abstract 2841: A co-clinical assessment of patterns of BRAF inhibitor resistance
Authors: Kwong Lawrence N. , Boland Genevieve , Frederick Dennie , Helms Timothy , Akid Ahmad , Miller John , Jiang Shan , Cooper Zachary , Song Xingzhi , Seth Sahil , Kamara Jennifer , Protopopov Alexei , Mills Gordon , Flaherty Keith , Wargo Jennifer , Chin Lynda
Published: August 2015

Multiple mechanisms conferring resistance of melanomas to BRAF inhibitors have been described, yet the basis of resistance in a sizeable portion of patient samples remains undefined. To address this gap, we characterized patients’ baseline, on-treatment, and resistant tumors on both the protein and RNA levels. By RNA-sequencing, we identified known resistance-conferring mutations in 50% (6/12) of the resistant samples. In parallel, targeted proteomic analysis by protein array categorized the resistant samples into three stable groups: two that reactivate MAPK signaling to different levels, and one that is MAPK-independent. The molecular relevance of these categories was supported by both mutation data and the similarity of resistance patterns that emerged in a co-clinical trial in a novel genetically engineered mouse model. Additionally, we have defined biomarkers in pre- and early-treatment patient samples that have potential for predicting clinical responses. On the basis of these observations, we suggest that BRAF inhibitor-resistant melanomas can be actionably categorized using protein patterns, even without the identification of the underlying genetic alteration.

Citation Format: Lawrence N. Kwong, Genevieve Boland, Dennie Frederick, Timothy Helms, Ahmad Akid, John Miller, Shan Jiang, Zachary Cooper, Xingzhi Song, Sahil Seth, Jennifer Kamara, Alexei Protopopov, Gordon Mills, Keith Flaherty, Jennifer Wargo, Lynda Chin. A co-clinical assessment of patterns of BRAF inhibitor resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2015-2841
Abstract 2849: The combination of the PARP inhibitor talazoparib (BMN 673) with the ATR inhibitor VE-821 overcomes the drug resistance of Schlafen 11-deficient cells
Authors: Murai Junko , Pommier Yves
Published: August 2015

Poly(ADP-ribose)polymerases (PARP) inhibitors work as catalytic inhibitors as well as PARP poisons by trapping PARP-DNA complexes (Murai et al., Cancer Res. 2012: 72:5588-99), and talazoparib is the most potent PARP inhibitor to date in terms of cytotoxicity and PARP trapping (Murai et al., Mol. Cancer Ther. 2014:13:433-43). Nevertheless, mining the NCI60 data shows that half of the 60 cell lines are tolerant to talazoparib. To elucidate the mechanisms of such tolerance, we searched for genes with expression levels correlated to talazoparib sensitivity using CellMiner (http://discover.nci.nih.gov/cellminer/). Schlafen 11 (SLFN11) turned out to be one of the most positively correlated genes whose expression is correlated with cellular sensitivity to talazoparib. Using genetically altered cells, we established that SLFN11 inactivation confers high resistance to talazoparib, revealing that SLFN11 inactivation, which is common in cancer cells (Zoppoli et al. PNAS 2014: 109:15030-5), is a novel mechanism of resistance for PARP inhibitors. Based on genetically engineered CRISPR/casp9 SLFN11-knockout cell lines and cell cycle analyses, we found that the parent SLFN11-proficient cells arrest in S phase without DNA replication, while SLFN11-knock out cells maintain their replication until entering G2 phase in the presence of talazoparib. Although ATR is the key regulator of intra-S phase check point, the addition of the ATR inhibitor (VE-821) (Josse et al., Cancer Res. 2014) does not rescue the replication arrest in SLFN11-proficient cells, suggesting the existence of a SLFN11-dependent replication arrest mechanism independently of ATR. On the other hand, the addition of the ATR inhibitor in SLFN11-knock out cells alters the arresting point from G2 to S phase, and eventually increases dead cells. This is probably because the prolonged replication block leads to the collapse of replication forks, resulting in DNA double-strand breaks. Consistent to these findings, the combination of the ATR inhibitor with talazoparib synergistically sensitizes SLFN11-knock out cells more efficiently than the parent SLFN11-proficient cells. Together, these results demonstrate that the combination of the ATR inhibitor with talazoparib can overcome the resistance of SLFN11-deficient cells to talazoparib.

Citation Format: Junko Murai, Yves Pommier. The combination of the PARP inhibitor talazoparib (BMN 673) with the ATR inhibitor VE-821 overcomes the drug resistance of Schlafen 11-deficient cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2849. doi:10.1158/1538-7445.AM2015-2849
Abstract 2857: Metastatic site and response to pembrolizumab (anti-PD1 antibody) in melanoma
Authors: Tumeh* Paul Camille , Rosenblum* Michael , Handley Nathan , Tsai Katy , Rodriguez Robert Rodriguez S. , Khurana Niharika , Harview Christina , Spasic Marko , Sanchez Phillip J. , Chang Jeremy , Shintaku I. Peter , Taylor Emma , Chmielowski Bartosz , Grogan Tristan , Elashoff David A. , Pierce Robert H. , Daud Adil
Published: August 2015

Therapies that block the PD-1/PD-L1 axis have shown significant clinical activity in melanoma and other cancers. Recent evidence has shown that pre-existing CD8 T cells infiltrates at the invasive tumor margin of metastatic melanoma are associated with the presence of the PD-1/PD-L1 immune axis and may predict response to therapy. We examined the relationship between site of metastatic disease, local immune response, and treatment outcome in patients with advanced melanoma treated with pembrolizumab. We studied patients enrolled in the pembrolizumab Phase I clinical trial in the intention to treat (ITT, N = 112) and full analysis set (FAS) cohorts (N = 96). Using quantitative immunohistochemistry and multi-parameter flow cytometry we analyzed the local immune response in pre-treatment metastatic samples according to organ site of metastasis and treatment outcome. The overall response rate was 40% with a median progression-free survival (PFS) of 5.58 months. The presence of melanoma metastatic to the liver (liver metastasis group) had an objective response rate (ORR) of 16% and median PFS of 2.79 months. The presence of melanoma metastatic to the lung (lung metastasis group) had an ORR of 64% and median PFS of 14.48 months. Preliminary analysis of samples obtained from patients with liver metastases showed reduced tumor infiltrating CD8+ T cells (TILs), PD-1 expression, and tumor PD-L1 expression in distant metastases. Phenotypic and functional analysis of TILs showed reduced percentages of partially exhausted PD-1+CTLA-4+CD8+ cells, which correlated with progression and with liver metastasis. These cells were shown to make IFNγ but not TNF or IL-2. Additionally, the presence of liver metastasis in melanoma patients prior to starting anti-PD1 therapy correlated with treatment failure. Liver metastasis was associated with fewer PD-1+ CTLA-4+ TILs in distant tumors. These preliminary results warrant further investigation into determining whether site of metastasis modulates the systemic immune response to PD-1/PD-L1 blocking therapies.

Citation Format: Paul Camille Tumeh*, Michael Rosenblum*, Nathan Handley, Katy Tsai, Robert Rodriguez S. Rodriguez, Niharika Khurana, Christina Harview, Marko Spasic, Phillip J. Sanchez, Jeremy Chang, I. Peter Shintaku, Emma Taylor, Bartosz Chmielowski, Tristan Grogan, David A. Elashoff, Robert H. Pierce, Adil Daud. Metastatic site and response to pembrolizumab (anti-PD1 antibody) in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2857. doi:10.1158/1538-7445.AM2015-2857
Abstract 2868: SETDB1 accelerates non-small cell lung cancer (NSCLC) tumorigenesis through WNT signalling pathway
Authors: Sun Qiaoyang , Ding Lingwen , Xiao Jinfen , Goodglick Lee , Chia David , Mah Vei , Alavi Mohammad , Doan Ngan , Said Jonathan W. , Yang Henry , Koeffler H.Phillip
Published: August 2015

Histone methylation deregulation leads to disruption of chromatin structure and gene expression pattern in many human cancers. Histone H3 lysine K9 methyltransferase, SETDB1 (SET domain, bifurcated 1) is initially reported to control heterochromatin structure, repress gene expression and be essential in early embryogenesis. Studies have recently shown its involvement in boosting tumorigenesis in different kinds of human cancers, including lung cancer. However, the mechanism underlying its ability to promote tumorigenesis remains largely unknown. In our study, we sought to investigate the oncogenic role of SETDB1 in NSCLC. Our immunohistochemistry found that 72% out of 387-lung cancer cases stained positively for SETDB1, compared to 46% samples of normal bronchial epithelium (p<0.0001). Moreover, the percent positive cells and their intensity of positivity correlated with cancer grades.

Ectopic expression of SETDB1 in NSCLC cell lines enhanced clonogenic growth in vitro and significantly enlarges tumor size in vivo; while depleting endogenous SETDB1 expression with shRNAs suppressed NSCLC cells proliferation and tumor formation. Analyzing the expression pattern of cells with forced expression of SETDB1 combined with TOP/FOP luciferase assay and western blotting showed that WNT/β-catenin signaling was activated upon overexpression of SETDB1, as well as upregulation of its downstream targets, c-Myc and Cyclin D1. ChIP assay showed that SETDB1 binds to WNT relevant genes, such as APOE, IGFBP4, FZD1 and LRP8 and upregulates their transcription, which is very distinct from its canonical suppressing function of H3K9 trimethylation. Interestingly, we also noted that SETDB1 was able to repress P53 expression, while diminishing P53 expression reciprocally enhanced SETDB1 expression.

In summary, our study demonstrates that SETDB1 enhances NSCLC tumorigenesis through activating WNT/β-catenin signaling and repressing tumor suppressor P53. Our novel findings provide new insight into a non-canonical function of SETDB1 in activating gene expression, and suggest therapeutic benefit of targeting SETDB1 in NSCLC having high expression level of this protein.

Citation Format: Qiaoyang Sun, Lingwen Ding, Jinfen Xiao, Lee Goodglick, David Chia, Vei Mah, Mohammad Alavi, Ngan Doan, Jonathan W. Said, Henry Yang, H.Phillip Koeffler. SETDB1 accelerates non-small cell lung cancer (NSCLC) tumorigenesis through WNT signalling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2868. doi:10.1158/1538-7445.AM2015-2868
Abstract 287: Specific delivery of immunostimulatory RNA via nanoparticles blocks growth of primary and disseminated ovarian tumors
Authors: Primiano Thomas , Chang Bey-ih
Published: August 2015

One important mechanism of immune evasion is the accumulation of tumor-infiltrating regulatory T cells creating an immunosuppressive microenvironment in situ. Many new therapies target immune checkpoint receptors, CTLA-4 and PD-1, on immunosuppressive T-cells to reverse tumor immune evasion. Small interfering RNAs (siRNAs) are double stranded, sequence-specific inhibitors of gene expression. The paradigm for using siRNA to block cancer is to target mRNA sequences of oncogenes. Several limitations have suppressed the use of siRNA in human cancer therapy, such as off-target effects and lack of tumor-specific delivery. Additionally, certain siRNA sequence motifs activate toll-like receptors that induce the systemic innate immune response, including induction of interferon and interleukin-6 release that is traditionally considered as an undesirable clinical outcome. The hypothesis proposed here is to control the innate immune response by directing it only to tumors and not other organs by delivering an immunostimulatory siRNA (isRNA) directly to tumors. An isRNA designed to silence no human genes was synthesized as an unmodified triphosphate 19-mer double-stranded RNA with a GUGA motif on the 5′ antisense end. This isRNA was delivered into human ovarian tumors grown from SK-OV-3 cells surgically implanted into the ovaries of immunosuppressed mice by an L1CAM-targeted elastin-like polypeptide (ELP-L) nanoparticle. The isRNA/ELP-L nanocomplex was dosed into ten mice by i.p. injection 14 times at 10 mg/kg with respect to the isRNA over 28 days. The isRNA was delivered to the tumors, not to liver, spleen, heart, or lungs. The total tumor mass and number of disseminated nodules both decreased by 50% with respect to vehicle control. Six ovaries harboring tumors were resected from isRNA/ELP-L nanocomplex-dosed mice and each displayed elevated levels of interferon alpha and interleukin 6 mRNA as determined by RT-PCR. Mean AST, ALT and BUN serum biomarkers of liver and kidney function were elevated by 68%, 128% and 14%, respectively, compared to vehicle dosed mice; none of which were outside average normal levels. These results indicate that specifically targeting isRNA to tumors using a targeted nanoparticle may provide an effective therapy for ovarian cancer, either alone or in combination with agents that affect immune check point pathways in tumors.

Citation Format: Thomas Primiano, Bey-ih Chang. Specific delivery of immunostimulatory RNA via nanoparticles blocks growth of primary and disseminated ovarian tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 287. doi:10.1158/1538-7445.AM2015-287
Abstract 2871: Allele-specific modulation of cancer metabolism by a long noncoding RNA
Authors: Redis Roxana S. , Ivan Cristina , Vela Luz , Lu Weiqin , Rodriguez-Aguayo Cristian , Ambrosio Andre LB , Gomes Dias Sandra M. , Berindan-Neagoe Ioana , Calin George A.
Published: August 2015

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the two major nutrients that fuel cellular metabolism and the pathways utilizing these nutrients are often altered in cancer. In this study we show that the long non-codingRNA CCAT2, located at 8q24 amplicon on cancer risk associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo, in an allele-specific manner by binding the cleavage factor I (CFIm) complex with distinct affinities, complementary to the risk for colon cancer. The newly formed RNA:protein complex, CCAT2:CFIm, regulates the alternative splicing of glutaminase 1 (GLS1) by selecting the poly(A) site in intron 14 of the precursor mRNA. This results in the preferential expression of the GAC, the more catalytically active GLS1 isoform. Our findings, supported by data in human colorectal cancer samples from multiple cohorts, including TCGA dataset, and multiple animal models, uncover complex mechanisms of cancer metabolism regulation controlled by a long non-codingRNA.

Citation Format: Roxana S. Redis, Cristina Ivan, Luz Vela, Weiqin Lu, Cristian Rodriguez-Aguayo, Andre LB Ambrosio, Sandra M. Gomes Dias, Ioana Berindan-Neagoe, George A. Calin. Allele-specific modulation of cancer metabolism by a long noncoding RNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2871. doi:10.1158/1538-7445.AM2015-2871
Abstract 2901: The role of basal and BRAF inhibitor-induced autophagy-driven secretion in remodeling the melanoma tumor secretome
Authors: Kraya Adam , Speicher David W. , Amaravadi Ravi
Published: August 2015

Autophagy is a homeostatic process of cellular self-digestion and is an adaptive therapy resistance mechanism in cancer. Emerging studies indicate that autophagy is involved in secretion through non-classical mechanisms. It is not well understood, however, how this secretory function modifies the molecular composition of the tumor secretome, which plays an important role in tumor growth and tumor immune responses. In this work, we focused on the role of both basal and BRAF inhibitor (BRAFi)-induced autophagy in melanoma cells harboring BRAF V600E, a missense mutation observed in ∼60% of melanoma tumors. Using quantitative proteomics we previously showed that a small number of proteins were differentially secreted by low and high autophagy melanoma cells grown in three-dimensional culture. Secreted proteins associated with high autophagy included IL-1β, IL-8, LIF, FAM3C, and DKK3, which have known roles in tumorigenesis. Further, serum collected from untreated metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from low autophagy patients. In the current study, melanoma cells with high autophagy and the BRAF V600E mutation (1205Lu) were treated with the BRAF inhibitor PLX4720, the autophagy inhibitor hydroxychloroquine (HCQ), PLX4720+HCQ, and the autophagy inducer rapamycin. The increase in autophagic flux upon the addition of PLX4720 or rapamycin was confirmed by western blotting and tandem fluorescently tagged LC3 (mCherry-eGFP-LC3). Secretomes were compared using quantitative proteomics, which resulted in identification of 1154 proteins with a false discovery rate of less than 1%. Hierarchical clustering and principal components analysis showed that secretomes from cells treated with the autophagy inducers rapamycin or PLX4720 correlated more closely with each other than those treated with HCQ either alone or in combination. A panel of proteins involved in extracellular matrix modification, cell-cell adhesion, and inflammation (including FAM3C and LIF) significantly increased in the supernatants of cells treated with PLX4720 or rapamycin and decreased in supernatants from cells treated with HCQ or HCQ+PLX4720. These results demonstrate that autophagy plays an active role in modifying the molecular composition of the tumor secretome. Proteins specifically shed in response to BRAF inhibitors and/or autophagy inhibition may have utility in the clinic for diagnostic and pharmacodynamic applications.

Citation Format: Adam Kraya, David W. Speicher, Ravi Amaravadi. The role of basal and BRAF inhibitor-induced autophagy-driven secretion in remodeling the melanoma tumor secretome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2901. doi:10.1158/1538-7445.AM2015-2901
Abstract 2906: Role of autophagy in Wnt5A-mediated melanoma invasion and metastasis
Authors: Ndoye Abibatou , Budina Anna , Webster Marie , Kaur Amanpreet , Behera Reeti , Murphy Maureen , Weeraratna Ashani T.
Published: August 2015

Melanoma is the most aggressive type of skin cancer and the leading cause of death from skin disease. With increasing incidence of the disease, it is crucial to further investigate the cellular mechanisms and molecular pathways that lead to invasion, metastasis, and drug resistance. Recent studies have revealed that high autophagy correlates with melanoma tumor aggressiveness and poor survival in clinical samples, as it is a common mechanism of resistance to therapy. Autophagy inhibition leads to reduced levels in Wnt5A in a breast cancer model, suggesting a cross-talk of Wnt5A and autophagy in cancer. β catenin, a mediator of canonical Wnt signaling, has been shown to act as a negative regulator of both basal and induced autophagy in a colorectal cancer model. Our laboratory showed that high levels of Wnt5A correlate with increased invasion and metastasis in melanoma and that Wnt5A downregulates β catenin; therefore, we hypothesize that autophagy might drive invasion and metastasis in aggressive melanoma through the regulation of Wnt signaling. To study the role of autophagy in Wnt5A-mediated melanoma invasion, we inhibited autophagy in highly invasive melanoma cells using lentivirus-mediated shATG5 knockdown or hydroxychloroquine and evaluated the effects of autophagy inhibition on Wnt5A expression, β catenin, and invasion using 3D spheroid models. Analysis of autophagy flux confirms that highly invasive melanoma cells have high autophagy compared to non-invasive cell lines. The inhibition of autophagy by hydroxychloroquine or shATG5 both resulted in significant decrease in invasion. By western blot analysis, we also observed a decrease in Wnt5A in these cells. These results demonstrate that autophagy inhibition in highly invasive melanoma leads to decrease in invasion in a 3D model that mimics the tumor microenvironment of melanoma. Further dissection of the molecular mechanisms that are involved in this process will enable the identification of novel autophagy targets in aggressive melanoma.

Citation Format: Abibatou Ndoye, Anna Budina, Marie Webster, Amanpreet Kaur, Reeti Behera, Maureen Murphy, Ashani T. Weeraratna. Role of autophagy in Wnt5A-mediated melanoma invasion and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2906. doi:10.1158/1538-7445.AM2015-2906
Abstract 2908: A sensitive immunohistochemistry method for in vivo autophagy biomarker detection and pharmacodynamic studies
Authors: Yang Yu , D'Amore Natalie Roy , Chouitar Jouhara , Yu Jie , Burke Kristine , Xiang Zhongmin , Yu Lunyin , Lasky Kerri , Brownell James , Simpson Chris , Luongo Evan , Bowman Doug , Tirrell Stephen , He Helen
Published: August 2015

Immunohistochemistry (IHC) is a powerful tool for studying pharmacodynamic biomarkers in tissue samples. However, IHC using standard methodologies to monitor autophagy has met with limited success due to the technical difficulties involved in detecting transient and low abundance autophagy markers. To address this issue, we have applied a highly sensitive IHC method using a tyramide-based signal amplification system to detect autophagy pathway proteins in paraffin sections of human tumor cell line and xenograft tissues. We were able to detect basal levels of punctate staining LC3B and NBR1 on both HCT116 and Calu-6 xenograft tumors using the tyramide-based signal amplification method whereas standard IHC showed little to no specific staining. Consistent with in vitro observations, lipidated LC3B, as detected by puncta formation, was elevated following autophagy induction using mTOR inhibitor treatment, and decreased following ATG7 knockout or knock down in both cell lines and xenograft tumors. As a result of autophagy pathway interruption following ATG7 inhibition, accumulation of the adapter proteins NBR1 and p62 was also detected. Our data demonstrate the feasibility of using high sensitivity IHC assays to reliably measure low abundant autophagy markers in xenograft tissues and may aid the exploration of autophagy regulation in cancer development.

Citation Format: Yu Yang, Natalie Roy D'Amore, Jouhara Chouitar, Jie Yu, Kristine Burke, Zhongmin Xiang, Lunyin Yu, Kerri Lasky, James Brownell, Chris Simpson, Evan Luongo, Doug Bowman, Stephen Tirrell, Helen He. A sensitive immunohistochemistry method for in vivo autophagy biomarker detection and pharmacodynamic studies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2908. doi:10.1158/1538-7445.AM2015-2908
Abstract 2914: Gold magneto nanoparticles induced apoptosis in liver cancer cells through changes of Ca channel pump detected by LIBS Technique and RT-PCR array
Authors: Ahmed Ola , Imam Hisham , Zekri Abdel-Rahman N.
Published: August 2015

Laser-induced breakdown spectroscopy technique (LIBS) was used in thesis study to address the effect of gold magneto nanoparticles on the liver cancer cells (HepG2) by examining the variation in genotoxicity and cytotoxicity.

HepG2 cell line was cultured at 105 cells/ml and expanded, after which, they were incubated with gold magneto nanoparticles at concentrations 10, 25, 50 μg/ml respectively for 12 hours. The effect of these nanoparticles in cell apoptosis as well as in autophagy was evaluated by acridine orange staining and Annexin V/propidium iodide double staining as well as apoptosis specific PCR array assay for evaluation of apoptosis in the treated cells.

At a concentration of nanocomposite 10, 25, 50 μg/ml, cells showed increase in the apoptosis figure from 11.25% ± 3.1% to 28% ± 2.04%, 39% ± 3.6%, 60% ± 4.3% respectively. Apoptosis specific PCR array also, showed very high expression of TNF, BIRC3 and NFKB1 with increasing in the fold regulation ratio of 155.03,158.03 and 149.2- fold respectively.

On the other hand, extra investigation of these treated cells by LIBS shows an increasing in the iron oxide nanoparticles concentration as well as calcium concentration. This increase in the calcium content in the treated cells may play an important role in the program cell apoptosis which was prove in our study by both Flow cytometry as well as by profile apoptosis PCR array.

Finally, our data showed the possibility of the using LIBS as inductor of cells apoptosis by measurement Ca concentration changes in liver cancer cells (HepG2 cells).

Keywords: Apaptosis, LIBS; HepG2 cell; Genotoxicity

Citation Format: Ola Ahmed, Hisham Imam, Abdel-Rahman N. Zekri. Gold magneto nanoparticles induced apoptosis in liver cancer cells through changes of Ca channel pump detected by LIBS Technique and RT-PCR array. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2914. doi:10.1158/1538-7445.AM2015-2914
Abstract 2935: A novel HSP70 inhibitor demonstrates potent anti-myeloma activity
Authors: Bailey Charvann K. , Budina Anna , Murphy Maureen , Nefedova Yulia
Published: August 2015

Multiple myeloma (MM) is a hematological cancer characterized by clonal proliferation of malignant plasma cells that produce monoclonal immunoglobulin. Because they secrete excess amounts of immunoglobulins, MM cells are typically under proteotoxic stress and utilize multiple mechanisms including molecular chaperones to maintain protein homeostasis. The chaperone HSP70 (heat shock protein 70) is overexpressed in MM. Therefore, we sought to evaluate whether targeting HSP70 with a novel HSP70 inhibitor would have a therapeutic benefit in MM. Toward this end, a panel of human MM cells lines including H929, RPMI-8226, MY-5, U266 and MM1s were tested for the IC50 to PET-16, an HSP70 inhibitor. Treatment with PET-16 resulted in a significant decrease in the viability of all MM cells tested with an IC50 value range from 3.5-7.2uM as was determined by the Alamar Blue assay. Decreased MM cell viability after treatment with PET-16 was due to induction of cellular apoptosis as was determined by Annexin V binding assay using flow cytometry. Because MM cells typically exist under hypoxic conditions, and hypoxia is known to decrease the efficacy of some chemotherapeutic agents, we analyzed the efficacy of PET-16 under hypoxia (1% oxygen). There was no difference in anti-MM effect of PET-16 in cells cultured under hypoxia as compared to those cultured in normoxic conditions. Taken together, our data suggest that targeting HSP70 with the novel HSP70 inhibitor PET-16 could be a viable potential strategy for treatment of MM.

Citation Format: Charvann K. Bailey, Anna Budina, Maureen Murphy, Yulia Nefedova. A novel HSP70 inhibitor demonstrates potent anti-myeloma activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2935. doi:10.1158/1538-7445.AM2015-2935
Abstract 2957: Investigating methyl-CpG DNA recognition in cancer
Authors: Terooatea Tommy W. , Wallace Megan J. , Szulik Marta W. , Chugg Alan C. , Miller Sven , Buck-Koehntop Bethany A.
Published: August 2015

DNA methylation is an essential epigenetic modification in eukaryotes required for genomic stability, control of gene expression and regulation of chromatin structure. Consequently, abhorrent alterations in methyl-CpG modifications across the genome, leading to inappropriate transcription, are associated with many diseases including cancer. Thus, an interest in discerning the regulatory mechanisms linking DNA methylation with gene expression in the disease state has emerged. The ZBTB methyl-CpG binding proteins (MBPs) are specialized transcription factors that mediate transcription by specifically targeting both methylated and sequence-specific DNA sites through a conserved set of Cys2His2 zinc fingers. While ZBTB MBP expression has become increasingly associated with higher grade tumors, we have only begun to understand the underlying complexities by which this family mediates gene expression. Here we combine interdisciplinary in cell genomic with in vitro biophysical approaches to begin to delineate the mechanisms by which this family of MBPs recognize DNA and regulate transcription in cancer. Specifically, for the founding member of this MBP family we utilized multiple genome-wide methodologies to identify its genomic targets, the methylation status at these sites and the transcriptional effect of this protein in cancer cell lines. We observed that protein expression levels, gene occupations and transcriptional responses were variable between cancer phenotypes. Additionally, ZBTB depletion results in variable phenotypic alterations between cell types indicating that protein presence may provide a means for defining these pathological states. Further, we have designed multiple protein constructs around the zinc finger domains of the ZBTB MBPs. Biophysical characterization of the binding interactions of these protein regions with various epigenetically modified DNAs has provided preliminary understanding for the differential mode of DNA recognition exhibited by the ZBTB MBP family members. Together, these findings provide initial insights into the molecular basis by which these proteins preferentially recognize, interpret and translate epigenetic signals into transcriptional responses in cancer cells.

Citation Format: Tommy W. Terooatea, Megan J. Wallace, Marta W. Szulik, Alan C. Chugg, Sven Miller, Bethany A. Buck-Koehntop. Investigating methyl-CpG DNA recognition in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2957. doi:10.1158/1538-7445.AM2015-2957
Abstract 2970: Whole genome sequencing analysis of multiple liver cancer nodules for determination of causal events for multi-occurrence
Authors: Furuta Mayuko , Fujimoto Akihiro , Ueno Masaki , Hayami Shinya , Kawakami Yoshi-iku , Gotoh Kunihito , Tsunoda Tatsuhiko , Miyano Satoru , Ohdan Hideki , Chayama Kazuaki , Yamaue Hiroki , Nakagawa Hidewaki
Published: August 2015

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Despite the recent progress in diagnostic accuracy and therapeutic efficacy, its five-year survival rate is still low. Characteristically, HCC shows a high rate of intrahepatic metastasis (IM) or multi-centric (MC) occurrence within the liver, and discrimination of IM/MC is difficult and important for therapeutic decision. Previously we reported that there were no common somatic mutations between whole genomes of MC tumors, indicating that MC tumors independently developed in their genetic aspect (Nat Genet, 44: 760-764, 2012). Here we performed whole genome sequencing (WGS) analysis on 43 multiple liver cancer nodules derived from 20 patients, which were clinically defined as IM or MC occurrence. We first analyzed common and unique somatic mutations in each individual and divided the cases into IM or MC by using the information of common somatic mutations between the multiple nodules. Interestingly, the clinical and pathological diagnoses of MC/IM in five of 20 cases were not concordant with the judgments from WGS, indicating the usefulness of WGS for a precise IM/MC judgment. Analysis of common somatic alterations in IM tumors enabled to extract early and late event in hepatocarcinogenesis among a large number of somatic events in each cases, while MC pairs shared only a few numbers of common alterations, including false positives. Importantly, one somatic mutation in non-coding region was exceptionally detected commonly among MC tumor pairs from three cases. Sanger sequencing confirmed that this mutation was also commonly detected in 10.9% (35/321) of liver cancer samples somatically. These results indicate the significance of this function-unknown and recurrent mutation in liver cancer development. To illuminate common driver events in MC tumors, we analyzed all detectable genetic alterations, including copy number alterations and rearrangements, on multi-time point or MC tumors derived from same individuals and extracted significant genetic alterations from them, which can help the understanding of driver genetic alterations in liver cancers.

Citation Format: Mayuko Furuta, Akihiro Fujimoto, Masaki Ueno, Shinya Hayami, Yoshi-iku Kawakami, Kunihito Gotoh, Tatsuhiko Tsunoda, Satoru Miyano, Hideki Ohdan, Kazuaki Chayama, Hiroki Yamaue, Hidewaki Nakagawa. Whole genome sequencing analysis of multiple liver cancer nodules for determination of causal events for multi-occurrence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2015-2970
Abstract 2978: An integrated comparative analysis of TCGA lung adenocarcioma and lung squamous cell carcinoma copy number and RNA-Seq expression data
Authors: OHara Andrea J. , Keshavan Raja , Che Zhiwei , Shams Soheil
Published: August 2015

The Cancer Genome Atlas (TCGA) contains various types of genomic data from a wide variety of cancers, several of which affect the same tissue site. Here we analyze copy number and RNA-Seq data from TCGA for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Level 1 (raw) data, rather than Level 3 (segmented) data, from TCGA was used for an integrated analysis of copy number and gene expression profiles of the two cancers. Using the copy number and SNP- probes, re-processed tumor profiles were more consistent with a control set in terms of median number of copy number events, sample ploidy, and breakpoint genes than with the published “level 3” TCGA data. Probe-level data were analyzed using Nexus Copy Number SNP-FASST2 (a multi-state HMM algorithm that uses both SNP and copy number probes in making state assignments), with systematic correction applied to correct for GC biases. Additionally we performed manual baseline adjustment to correct for sample ploidy based on whole-genome B-alelle frequency data for each sample. Overall, the median number of copy number events in the LUAD TCGA data set was reduced from 371 (in the level 3 set) to 299, and from 681 (in the level 3 set) to 177 in the LUSC TCGA data set. After manual inspection, more than 38% of the TCGA LUAD samples and 50% of the TCGA LUSC samples available at level 3 were found to have incorrect baseline ploidy assignments. The resultant re-analyzed copy number data sets were used for an integrated analysis between the two tumor types. Comprehensive comparative analysis using Fisher's Exact Test revealed statistically significant differences (percent differential = 25%, p<0.001) in copy number profiles between the two lung tumor types; copy number changes include differential loss of chromosome 1p, loss of chromosome 3p, gain of chromosome 3q, loss of chromosome 4 and loss of chromosome 5. These correlated to changes in overall survival: individuals with loss of chromosome 1p and/or loss of 5q resulted in a significantly poorer prognosis (p<0.05). While more frequent in the LUSC sample population, this change in overall survival outcome was extended to samples with chromosome 1p loss in LUAD samples as well. Integration with RNA-Seq expression data from each tumor type revealed statistically significant correlations (p<0.05) with these copy number alterations, identifying potential driver genes of interest among each subtype and lung tumors in general.

Citation Format: Andrea J. OHara, Raja Keshavan, Zhiwei Che, Soheil Shams. An integrated comparative analysis of TCGA lung adenocarcioma and lung squamous cell carcinoma copy number and RNA-Seq expression data. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2978. doi:10.1158/1538-7445.AM2015-2978
Abstract 299: In vitro compound screening identifies enhancers of adenoviral oncolysis with Delta24-RGD in patient-derived glioblastoma stem cells
Authors: Berghauser Pont Lotte ME , Balvers Rutger , Kloezeman Jenneke , Nowicki Michal O. , Kremer Andreas , Chiocca E. Antonio , Leenstra Sieger , Dirven Clemens MF , Lawler Sean , Lamfers Martine LM
Published: August 2015

The tumor targeted oncolytic adenovirus Delta24-RGD is currently under phase I/II investigation for the malignant brain tumor glioblastoma. Despite encouraging results, the efficacy of oncolytic virotherapy still requires improvements due to heterogeneous or poor responses. In this study, we performed a screen of 446 clinically applied drugs to identify those that enhance Delta24-RGD oncolysis in glioblastoma. Cell viability was determined five days post-infection in Delta24-RGD resistant patient-derived glioblastoma stem cell (GSCs) cultures. Potential ‘hits’ were tested for synergistic viral sensitization using the Chou-Talalay method. Effects on viral infection and replication were investigated using Ad-Luc-RGD and Delta24-RGD-GFP viruses, and apoptosis and necrosis were evaluated using caspase-3/7 and lactate dehydrogenase (LDH) assays. Selection based on the efficacy of combination treatment led to the identification of ten drugs as potential Delta24-RGD sensitizers from the initial screen. Further analysis of effects on viral replication, synergistic interactions and ability to penetrate the blood-brain barrier narrowed this down to four remaining compounds, fluphenazine, indirubin, lofepramine and ranolazine. These four agents increased caspase-3/7 activity and fluphenazine also increased LDH levels in combination with Delta24-RGD. Fluphenazine, indirubin, lofepramine and ranolazine sensitized 12/12, 11/12, 9/12 and 11/12 distinct GSC cultures to Delta24-RGD, respectively. In conclusion, a clinical compound screen on glioblastoma stem cells in combination with in vitro mechanistic studies, revealed four highly effective compounds that sensitize GSCs to Delta24-RGD oncolytic therapy.

Citation Format: Lotte ME Berghauser Pont, Rutger Balvers, Jenneke Kloezeman, Michal O. Nowicki, Andreas Kremer, E. Antonio Chiocca, Sieger Leenstra, Clemens MF Dirven, Sean Lawler, Martine LM Lamfers. In vitro compound screening identifies enhancers of adenoviral oncolysis with Delta24-RGD in patient-derived glioblastoma stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2015-299