bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3996: Integrated analysis of miRNA profiling and bioinformatics reveals the potential key miRNAs in gastric cancer
Authors: Zhang Xiaojing , Feng Xianling , Peng Yin , Yang Mengting , Huang Yong , Fan Xinmin , Meltzer Stephen J. , Jin Zhe
Published: August 2015

Supported by: National Natural Science Foundation of China, No.81172282; National Natural Youth Science Foundation of China, No.81302151; Shenzhen Peacock Plan, No.KQCX20130621101141669; Planned Science and Technology Project of Shenzhen, No.GJHS20120621142654087; the Key Laboratory Project of Shenzhen, No.ZDSY20130329101130496; Technological innovation group of SZU grants, No.T201202; NIH grants DK087454, CA146799, CA173390; an American Cancer Society Clinical Research Professorship.

Gastric cancer (GC) remains a major health threat worldwide, and is one of the leading causes of cancer-related deaths in China. The discovery of microRNAs (miRNAs) has provided a new and powerful tool for studying diagnostic biomarkers and effective therapeutic targets in GC. By using microarray analyses of benign and malignant gastric epithelial cell lines (HFE145, NCI-N87, MKN28, RF1, KATO III and RF48), we discovered dysregulated 16 miRNAs, of which 11 were validated by real-time qRT-PCR. Based on miRWalk online database scans, 2532 potential mRNA targets of these 16 miRNAs were identified. Bioinformatic analyses suggested that these predicted targets were principally involved in tumor pathogenesis, MAPK signaling, and apoptosis. Finally, miRNA-gene network analyses identified miRNA- 125b as being potentially vitally important in GC.

Citation Format: Xiaojing Zhang, Xianling Feng, Yin Peng, Mengting Yang, Yong Huang, Xinmin Fan, Stephen J. Meltzer, Zhe Jin. Integrated analysis of miRNA profiling and bioinformatics reveals the potential key miRNAs in gastric 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 3996. doi:10.1158/1538-7445.AM2015-3996
Abstract 4: ABT-263 is effective in a subset of non-small cell lung cancer cell lines
Authors: Kuroda Aoi , Ohgino Keiko , Yasuda Hiroyuki , Hamamoto Junko , Arai Daisuke , Ishioka Kota , Tani Tetsuo , Nukaga Shigenari , Kawada Ichiro , Naoki Katsuhiko , Soejima Kenzo , Betsuyaku Tomoko
Published: August 2015

Rationale:

ABT-263 (Navitoclax) is one of the BH3 mimetics targeting anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins such as Bcl-2, Bcl-XL, and Bcl-w, thereby inducing apoptosis. It has been reported that the response to ABT-263 is associated with expressions of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein. Given its effectiveness as a single agent in preclinical studies, ABT-263 is currently being evaluated in clinical trials for small cell lung cancer (SCLC) and leukemia. However, the efficacy of ABT-263 in non-small cell lung cancer (NSCLC) has not been fully evaluated. We examined the effect of ABT-263 on cell proliferation of NSCLC cell lines and investigated the underlying mechanisms.

Methods:

The following 9 NSCLC cell lines were examined: SK-LU-1, A549, H358, Calu3, H3122, H1975, H460, H441, and BID007. The effects of ABT-263 in NSCLC cell lines were evaluated by MTS assay. Apoptosis was examined by flowcytometry using staining for annexin V and propidium iodide (PI), and also western blotting for cleaved PARP. Quantitative RT-PCR was carried out to assess the mRNA expression levels of anti-apoptotic genes and pro-apoptotic genes. Immunoprecipitation and western blotting were performed to compare the levels of anti-apoptotic and pro-apoptotic proteins between the sensitive and resistant cell lines. In addition, knockdown of Mcl-1 was performed by siRNA.

Results:

By screening 9 NSCLC cell lines using MTS assay, we found Calu3 and BID007were sensitive to ABT-263. We also confirmed that apoptosis was induced only in the ABT-263 sensitive lines but not in the ABT-263 resistant cell lines after ABT-263 treatment. However, the expression levels of Bcl-2 family proteins, including Mcl-1, did not differ significantly among the ABT-263 sensitive and resistant cell lines. Unlike the results in previous reports regarding SCLC, Mcl-1 was not decreased in the sensitive cell lines. The ABT-263 resistant cell lines became sensitive to ABT-263 after knockdown of Mcl-1 by siRNA, while the ABT-263 sensitive cell lines maintained the same sensitivity.

Conclusion:

We found that Calu3 and BID007 were sensitive to ABT-263. In the sensitive NSCLC cell lines, ABT-263 induces apoptosis irrespective of Mcl-1 expression levels.

Citation Format: Aoi Kuroda, Keiko Ohgino, Hiroyuki Yasuda, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Tetsuo Tani, Shigenari Nukaga, Ichiro Kawada, Katsuhiko Naoki, Kenzo Soejima, Tomoko Betsuyaku. ABT-263 is effective in a subset of non-small cell lung cancer cell lines. [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 4. doi:10.1158/1538-7445.AM2015-4
Abstract 4000: The increase of oncogenic miRNA expression in tongue carcinogenesis of a mouse model
Authors: KAO YUYU , Lin Shu-Chun , Chang Kuo-Wei
Published: August 2015

Oral carcinoma is one of the leading causes of cancer death in the world. To develop biomarkers for early detection may bestow substantial benefits in diagnosis and the prevention of tumor progression. An animal model simulating the human pathogenesis will also be useful for developing therapeutic strategies. miRNAs are short, non-coding RNAs, which play important roles in neoplastic process. The detection of biomarkers in body fluid may enable non-invasive diagnostic approaches. This study used mouse chemical carcinogenesis model to investigate the disruption of miRNA expression in oral tongue tissues, and to explore the miRNA level in saliva and blood during pathogenesis. Mice were fed with 4-Nitroquinoline 1-Oxide (4NQO) in drinking water for 12 weeks and were kept for extended periods for tumor induction. The histological examination showed the consecutive increase in the severity of epithelial pathogenesis from hyperkeratosis, hyperplasia to epithelial dysplasia and squamous tumors following the increase of 4NQO exposure. The increase of miR-21 and miR-31 staining, as well as p53, pAKT and Ki-67 immunoreactivity parallels the severity of epithelial pathogenesis in tongue tissues. A progressive increase of multiple oncogenic miRNAs in both saliva and blood samples was also noted. The increase of miR-21, miR-31 and miR-372 in the saliva and the increase of miR-146a and miR-184 in the blood sample were particularly eminent during the field cancerization process of tongue. In addition, the dysregulation of these oncogenic miRNAs and other molecular events in this model are similar to the disruption being identified in human counterparts. As these miRNAs are functionally oncogenic, their increase in the saliva and blood of mice may be valuable biomarkers to develop diagnosis and prevention strategies against oral carcinomas.

Keywords: chemical, carcinoma, miRNA, mouse, mouth

Note: This abstract was not presented at the meeting.

Citation Format: YUYU KAO, Shu-Chun Lin, Kuo-Wei Chang. The increase of oncogenic miRNA expression in tongue carcinogenesis of a mouse model. [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 4000. doi:10.1158/1538-7445.AM2015-4000
Abstract 4010: Prognostic role of miRNAs in resectable non-small cell lung cancer
Authors: Gallach Sandra , Jantus-Lewintre Eloisa , Calabuig-Fariariñas Silvia , Blasco Ana , Guijarro Ricardo , Martorell Miguel , Camps Carlos
Published: August 2015

BACKGROUND MicroRNAs (miRNA) are a class of small RNAs that regulate gene expression, and are implicated in several diseases including cancer. The aim of this study was to analyze the prognostic implications of deregulated miRNAs identified by NGS in a previous study performed on resectable-NSCLC tumor samples.

MATERIAL AND METHODS Fresh-frozen tumor and normal adjacent lung tissue samples (N = 195) were obtained by surgical resection from NSCLC patients and storage in RNAlater® at -80°C until analysis. RNA was isolated biological specimens by Trizol based-methods. 22 deregulated miRNA were analyzed in paired tumor/normal samples by RTqPCR using TaqMan® microRNA assays. Statistical analyses were considered significant at p<0.05.

RESULTS Patient's median age was 64 years [26-85], 88.2% were male, 66.7% had good performance status (PS = 0), 39.5% were adenocarcinomas and 54.9% presented stage I at the time of the diagnosis. For survival analysis, continuous variables were dichotomized as high (≥ median) and low (< median), using the median expression of each miRNA as a cutoff. Of the 22 miRNAs analyzed only miR-21 and miR-188-5p had prognostic value. We found that patients with high levels of miR-21 and miR-188-5p had a significant reduction in PFS and OS rates. Interestingly, the group of patients with the combined high expression of both miRNAs (miR-21high & miR-188-5phigh) showed shorter PFS and OS (Table 1). Clinico-pathological and analytical variables were included in the multivariate analysis revealing that combined miRNAs is an independent prognostic marker for PFS (HR 0.485 [0.313-0.753]; p = 0.001) and OS (HR 0.389 [0.237-0.638]; p<0.0001].

a P value with Bonferroni correction are shown in parenthesesOSOSPFSPFSMedian (months)PaMedian (months)PamiR-21 (High vs low)42.60 vs 82.600.01622.13 vs 66.970.017miR-188-5p (High vs low)46.67 vs NR0.00524.03 vs 66.970.023Combined miRNAs (miR21 high miR-188 high vs combonations)21.13 vs 83.570.002 (0.008)16.97 vs 56.830.001 (0.004)

CONCLUSION Expression of miR-21 and miR-188-5p seems to have a prognostic role in NSCLC, and the combination of both could be an independent prognostic marker in early-stage NSCLC.

Table 1. OS and PFS for miR-21 and miR-188-5p in NSCLC patients (univariate analysis) Supported by grants RTICC (RD12/0036/0025 and RD06/0020/1024), TRACE (TRA09-0132) and Beca Roche Oncohematología.

Citation Format: Sandra Gallach, Eloisa Jantus-Lewintre, Silvia Calabuig-Fariariñas, Ana Blasco, Ricardo Guijarro, Miguel Martorell, Carlos Camps. Prognostic role of miRNAs in resectable non-small cell lung 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 4010. doi:10.1158/1538-7445.AM2015-4010
Abstract 4022: Fibroblast-derived PEDF inhibits tumor growth and angiogenesis and is suppressed by TGF-β and PDGF
Authors: Nwani Nkechiyere G.
Published: August 2015

Rationale and Objective: PEDF (pigment epithelium-derived factor) is an endogenous angiogenesis inhibitor and a type II tumor suppressor whose expression is often reduced/ lost in a variety of tumors, including malignant melanoma. To date, the mechanisms and consequences of PEDF loss on tumor progression have been investigated in neoplastic cells but not in the tumor-associated non-neoplastic cells. Fibroblasts are extremely abundant in the tumor microenvironment and can switch to the tumor-promoting phenotype in response to the tumor-associated factors. Here, we investigated how the loss of fibroblast-derived PEDF in the tumor microenvironment alters melanoma progression and the mechanisms, by which melanoma cells attenuate PEDF expression in fibroblasts.

Methods: We used lentiviral vectors for PEDF knockdown (shPEDF and control). Tumor growth was evaluated in immunodeficient mice (flank injections). Angiogenesis and proliferation in the tumor were measured by immunohistochemistry (IHC) for CD31 and Ki-67, respectively. Gene expression was evaluated by q-PCR and protein levels were detected by western blot/ IHC. The contribution of growth factors tested using specific pathway inhibitors.

Results: We have measured tumorigenesis of highly aggressive melanoma cell line C8161-HA co-mixed with shPEDF or control fibroblasts. PEDF loss in fibroblasts promoted tumor growth/ angiogenesis in xenografted human melanoma. Seeking mechanisms by which tumors attenuate PEDF expression in fibroblasts we found that it is mildly attenuated by hypoxia and potently down-regulated by the soluble factors produced by melanoma cells. Using recombinant growth factors and tumor conditioned media in combination with receptor and pathway-specific inhibitors we have identified TGF-β and PDGF as the main antagonists of PEDF expression. We have also discovered that PDGF directly blocks PEDF expression while regulation by TGF-β is indirect, whereby it elevates PDGFR expression by fibroblasts and thus increases their sensitivity to both paracrine and autocrine PDGF.

Conclusions: Together, our data demonstrate the key role of PEDF loss in non-neoplastic cells in the tumor microenvironment and the mechanisms by which tumors overcome its anti-cancer effects. Our data also indicate potential utility of PDGF inhibitors to restore PEDF in the tumor microenvironment.

Citation Format: Nkechiyere G. Nwani. Fibroblast-derived PEDF inhibits tumor growth and angiogenesis and is suppressed by TGF-β and PDGF. [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 4022. doi:10.1158/1538-7445.AM2015-4022
Abstract 4024: Understanding the role of mitochondria in the progression from normal fibroblasts to DCIS associated fibroblasts
Authors: Ko Ying-Hui , Lin Zhao , Martinez Outschoorn Ubaldo E.
Published: August 2015

Background and Purpose: Ductal carcinoma in situ (DCIS) is a non-invasive breast cancer which may progress to invasive breast cancer. Many studies have focused on the epithelial cell mechanisms of progression from normal breast tissue to DCIS. However, in this study we want to study mechanisms of progression in cancer associated fibroblasts from normal breast tissue to DCIS.

Loss of caveolin 1 (CAV1) in fibroblasts is a marker of reduced mitochondrial membrane potential and is a prognostic biomarker in ductal carcinoma in situ. High mitochondrial membrane potential in epithelial cells is a crucial determinant of carcinogenesis.

Methods: In the study we use MCF10DCIS as a model for DCIS. MCF10DCIS.com (MCF10DCIS) is a clonal breast cancer cell line. The MCF10A, non-tumorigenic epithelial cell line, and human immortalized but non-transformed dermal fibroblasts (BJ1) were used study the difference between non-tumorigenic epithelial cells and DCIS and interactions between these two epithelial cells and fibroblasts. In order to study the interaction between epithelial cells and fibroblasts, we set up a coculture model system comprised both epithelial cells and fibroblasts. Immunofluorescence staining and confocal microscopy were used here to visualize the specific protein expression in different cell types.

Results: (1) MCF10DCIS cells in coculture with fibroblasts have increased mitotracker red staining while as fibroblasts have decreased mitotracker staining compared to homotypic culture. However, coculture of MCF10A cells with fibroblasts did not change the mitotracker staining in either MCF10A or fibroblasts. (2) Loss of stromal CAV1 protein expression is observed in fibroblasts when fibroblasts are cocultured with MCF10DCIS cells compared to homotypic culture. CAV1 protein expression is unchanged in coculture with MCF10A cells.

Conclusion: Previous studies have demonstrated that loss of stromal CAV1 and changes in mitochondrial membrane potential are factors involved in the transition from DCIS to invasive breast cancer. In this study we show that loss of stromal CAV1 and increases in epithelial mitochondrial membrane potential may play a role in the progression to DCIS.

Citation Format: Ying-Hui Ko, Zhao Lin, Ubaldo E. Martinez Outschoorn. Understanding the role of mitochondria in the progression from normal fibroblasts to DCIS associated fibroblasts. [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 4024. doi:10.1158/1538-7445.AM2015-4024
Abstract 4036: Palmitoylated Ras-driven MAPK signaling, transformation and tumorigenesis, but not tumor progression, are spatially regulated by plasma membrane microdomains
Authors: Michael James , Wurtzel Jeremy G.T. , Goldfinger Lawrence E.
Published: August 2015

In this study, we assessed the contributions of plasma membrane (PM) microdomain targeting to the functions of H-Ras and paralogue R-Ras. Activated H-Ras is a mitogen which can drive tumorigenesis through Raf activation, while R-Ras is weakly mitogenic and does not induce the MAPK pathway in cells, despite binding Raf in vitro. These paralogues have identical effector-binding regions, but variant C-terminal targeting domains (tDs). Ras tDs are responsible for lateral distribution within PM microdomains as a result of distinct lipid modifications: activated H-Ras targets to lipid ordered/disordered (Lo/Ld) domain borders, whereas active R-Ras is sequestered in Lo domains (rafts). We hypothesize that PM segregation regulates effector interactions and downstream signaling. We used tD swap mutants, and assessed effects on MAPK pathway activation, cell proliferation, transformation, and tumorigenesis. Activated R-Ras harboring the H-Ras tD (R-Ras-tH), stably expressed in NIH3T3 fibroblasts, interacted with Raf and induced MEK and ERK phosphorylation. Furthermore, R-Ras-tH stimulated cell growth, and supported anchorage-independent growth in soft agar, indicating induction of a transformed phenotype in vitro. Conversely, stable transfectants of H-Ras harboring the R-Ras tD (H-Ras-tR) had reduced MEK and ERK phosphorylation, cell growth, and colony formation in soft agar compared with H-Ras cells. Thus, Ras access to Raf at the PM is a critical step for Ras mitogenic signaling. However, tumor progression driven by these cells in xenografts in mice did not match in vitro phenotypes. R-Ras-expressing cells produced few solid tumors, while R-Ras-tH cells produced relatively larger, but size-limited, avascular tumors with a higher frequency per xenograft. Surprisingly, both H-Ras and H-Ras-tR xenografts produced large and heavily vascularized tumors, implicating an important role of Ras in modulating tumor angiogenesis in this model. Indeed, the conditioned media (secretome) of R-Ras-expressing cells did not support endothelial cell cord formation in growth factor-reduced Matrigel, while the secretome from H-Ras cells robustly induced cord formation; these effects were independent of Ras microdomain localization. Thus, targeting Ras to the Lo/Ld border is sufficient to initiate tumor formation, however, a tD-independent angiogenic effect is required for progression of Ras-induced tumors.

Citation Format: James Michael, Jeremy G.T. Wurtzel, Lawrence E. Goldfinger. Palmitoylated Ras-driven MAPK signaling, transformation and tumorigenesis, but not tumor progression, are spatially regulated by plasma membrane microdomains. [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 4036. doi:10.1158/1538-7445.AM2015-4036
Abstract 4039: Inhibition of Pak4 blocks growth of breast cancer cells
Authors: Rane Chetan K. , Senapedis William , Baloglu Erkan , Shacham Sharon , Minden Audrey G.
Published: August 2015

The p21-activated kinase family of proteins promotes cell survival and plays an important role in cell proliferation, cell cycle regulation and cell shape determination. There are six mammalian PAK proteins which can be subdivided into two groups by sequence homology and mode of activation- Group A Paks consisting of Pak 1, 2 and 3 and Group B Paks consisting of Pak 4, 5 and 6. There is a growing list of evidence that PAK proteins are overexpressed in many cancer types, including colon, lung, and breast cancer. PAK4 is frequently overproduced in breast cancer, including Her2 positive and triple negative breast cancers, while it is expressed at low levels in normal mammary tissue. Our preliminary evidence showed that untransformed mouse mammary epithelial cells have low levels of PAK4 that become elevated when these cells are transformed by Her2 overexpression. Furthermore, PAK4 overexpressed in mammary epithelial cells leads to oncogenic transformation and tumorigenesis in mice. We hypothesize that PAK4 plays an important role in promoting mammary tumorigenesis, and that it can serve as an effective drug target for breast cancer treatment. To test this hypothesis, we have analyzed two orally available PAK4 Allosteric Modulators (PAM; KPT-8752 and KPT-9274), which reduce the steady state PAK4 protein level in cancer cells. We observed that when we treat breast cancer cells with KPT-8752, we severely block the cell growth. KPT-8752, is effective both in Her2 positive and triple negative breast cancer cells. Our results indicate that Pak4 is an important regulator of breast cancer cell growth. Experiments studying the effects of PAMs (KPT8752 and KPT9274) on tumorigenesis in animal models of breast cancer are ongoing.

Citation Format: Chetan K. Rane, William Senapedis, Erkan Baloglu, Sharon Shacham, Audrey G. Minden. Inhibition of Pak4 blocks growth of breast cancer 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 4039. doi:10.1158/1538-7445.AM2015-4039
Abstract 4042: Kruppel-like factor 5 promotes breast cancer proliferation, migration and invasion partially through upregulating the transcription of TNFα-induced protein 2
Authors: Jia Lin , Liu Rong , Chen Ceshi
Published: August 2015

The Krupple-like factor 5 (KLF5) transcription factor is highly expressed in high-grade and basal-like TNBC breast cancers. Our previous studies reported that KLF5 promotes breast cancer cell proliferation and survival through target genes, such as FGF-BP and mPGES1. However, the mechanism by which KLF5 promotes cell migration and invasion is still not completely understood. In this study, we demonstrated that TNFAIP2, a TNFα-induced gene, is a KLF5 direct downstream target gene. The expression of the TNFAIP2 is highly correlated with the expression of the KLF5 protein in breast epithelial cell lines at both mRNA and protein levels. Manipulation of KLF5 expression positively alters TNFAIP2 mRNA and protein expression levels in multiple breast cell lines. KLF5 binds to TNFAIP2 gene promoter and activates its transcription. Functionally, TNFAIP2 promotes cell proliferation and xenograft growth in NOD-SCID mice. Importantly, knockdown of either KLF5 or TNFAIP2 significantly reduces cancer cell migration and invasion. Overexpression of TNFAIP2 rescues KLF5 knockdown-induced migration suppression in HCC1937 cells. TNFAIP2 interacts with two small GTPase Cdc42 and Rac1, increases their activities, and changes actin skeleton and cell morphyology. Finally, KLF5 and TNFAIP2 promote cell migration and invasion through activating Cdc42 and Rac1. These findings collectively suggest that TNFAIP2 is a KLF5 direct target gene and both KLF5 and TNFAIP2 promotes TNBC cell migration and invasion through Cdc42 and Rac1.

Citation Format: Lin Jia, Rong Liu, Ceshi Chen. Kruppel-like factor 5 promotes breast cancer proliferation, migration and invasion partially through upregulating the transcription of TNFα-induced protein 2. [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 4042. doi:10.1158/1538-7445.AM2015-4042
Abstract 4056: Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses
Authors: IKUSHIMA Hiroaki , TANIGUCHI Tadatsugu
Published: August 2015

The eradication of cancer cells requires communication between cells which constitute the complex immune system. Natural killer (NK) cells are essential tumor-killing effector cells of the innate arm of the immune system; however, little is known about whether or how other immune cells recognize tumor cells to assist NK cells. In addition, although signal-transducing pattern recognition receptors are known to play important roles in innate immune responses against invading pathogens through recognition of pathogen-associated molecular patterns (PAMPs), it is still enigmatic whether and how these receptors contribute to anti-tumor immune responses. In this study, we show that the innate immune pattern recognition receptor Dectin-1 expressed on dendritic cells (DCs) and macrophages is critical to NK-mediated killing of tumor cells. We also show that tumor cell-mediated Dectin-1 signaling is instigated by the receptor recognition of N-glycan structures on tumor cells, which we term tumor-associated molecular patterns (TAMPs). As such, the TAMP-Dectin-1 signaling causes activation of the Interferon Regulatory Factor 5 (IRF5) transcription factor and subsequent induction of genes required for the full-blown killing activity of NK cells. The importance of these events is underscored by the observation of massive tumor metastasis in mice genetically deficient in either Dectin-1 or IRF5. Thus, these results reveal a hitherto unrecognized facet of pattern recognition receptors in the orchestration of anti-tumor innate immune responses; recognition of TAMPs. This study is the first demonstration that an innate immune pattern recognition receptor potentiates anti-tumor immune responses, offering new insight into anti-tumor activity of the innate immune system with implications for anti-tumor immunotherapy.

Citation Format: Hiroaki IKUSHIMA, Tadatsugu TANIGUCHI. Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses. [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 4056. doi:10.1158/1538-7445.AM2015-4056
Abstract 4058: Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism
Authors: Labiano Sara , Palazón Asís , Quetglas José I. , Bolaños Elixabet , Azpilicueta Arantza , Morales-Kastresana Aizea , Rodriguez Alfonso , Rodriguez-Ruiz Maria , Gurpide Alfonso , Aznar M Angela , Jure-Kunkel Maria , Melero Ignacio
Published: August 2015

Hypoxia is a common feature in solid tumors that has been implicated in immune-evasion. In this study, we show that exposure of mouse and human tumor cell lines to hypoxic conditions (1% O2) promotes CD137 transcription. Previous studies from our group have shown that hypoxia up-regulates the co-stimulatory receptor CD137 on activated T lymphocytes and on vascular endothelial cells. However, our results show that the CD137 transcript upregulated under hypoxia in tumor cell lines is predominantly an alternatively spliced form that encodes for a soluble variant, which lacks the transmembrane domain. Accordingly, soluble CD137 (sCD137) is detectable by ELISA in the supernatant of hypoxia-exposed cell lines and in the serum of tumor-bearing mice. sCD137, as secreted by tumor cells, is able to bind to CD137-Ligand (CD137L). Our studies on primed T lymphocytes in co-culture with stable transfectants for CD137L demonstrate that tumor-secreted sCD137 prevents co-stimulation of T lymphocytes. Such an effect results from preventing the interaction of CD137L with the transmembrane forms of CD137 expressed on T lymphocytes undergoing activation. This mechanism is interpreted as a molecular strategy deployed by tumors to repress lymphocyte co-stimulation via CD137/CD137L.

Citation Format: Sara Labiano, Asís Palazón, José I. Quetglas, Elixabet Bolaños, Arantza Azpilicueta, Aizea Morales-Kastresana, Alfonso Rodriguez, Maria Rodriguez-Ruiz, Alfonso Gurpide, M Angela Aznar, Maria Jure-Kunkel, Ignacio Melero. Hypoxia-induced soluble CD137 in malignant cells blocks CD137L-costimulation as an immune escape mechanism. [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 4058. doi:10.1158/1538-7445.AM2015-4058
Abstract 4059: Crosstalk between CD8+ T and NK cells: fine-tuning of antitumor immune response
Authors: Uzhachenko Roman V. , Goodwin J Shawn , Costa Lino , Terekhov Alexander , Thounaojam Menaka C. , Hofmeister William H. , Shanker Anil
Published: August 2015

Local tumor antigen-specific T cell-NK cell collaboration is indispensable for the elimination of tumor cells, including antigen-deficient tumor escape variants before metastasis. While mechanistic details are available for the innate instruction of the T cell responses, little is known for the adaptive control of NK cell activity. We observed in a mouse model of mastocytoma expressing a self tumor antigen P1A that effector CD8+ T cells provided a necessary “help” to dormant NK cells in eliciting their antitumor effector function. Bioluminescence imaging of mastocytoma tumors following adoptive transfer of P1A-specific T cells in RAG-/- and RAG-/-γc-/- mice showed that NK cell anti-tumor activity requires cytolytic T cells, whereas T cells can function independent of NK cells. In 2D and 3D co-culture systems, we observed that PMA/ionomycin-stimulated CD8+ T cells form multiple contacts with naïve NK lymphocytes. Data show that NK cells interacting with activated CD8+ T cells show an up-regulation of CD25 and CD69 expression mediated by intercellular contacts, and activation of NKG2D receptors and Stat2, Stat6, Jak1, Jak3, Tyk2, and PTEN signaling molecules with a decrease in the phosphorylation of Stat1, PKB/Akt, SAPK/JNK, p38. On the other hand, interacting NK cells down-regulate CD25 molecule expression on CD8+ T cells and promote differentiation of central memory CD44+CD62L+ T cells. CD8+ T cells display an elevation in the phosphorylation of Stat1 and down-regulation of Stat5 with stimulated PKB/Akt, Lck, mTOR, and p42/p44. Moreover, significant changes in the cytosolic and mitochondrial Ca2+, production of mitochondrial ROS, mitochondrial membrane potential, mitochondrial permeability transition pore, and synthesis of nitric oxide and non-protein thiols (mostly, reduced glutathion) were observed in a reciprocal T cell-NK cell interaction. These results highlight the importance of mitochondrial activity in the re-modeling of activation signaling and memory differentiation of interacting CD8 T cells and NK cells. These results will help refine cancer immunotherapeutic strategies.

Citation Format: Roman V. Uzhachenko, J Shawn Goodwin, Lino Costa, Alexander Terekhov, Menaka C. Thounaojam, William H. Hofmeister, Anil Shanker. Crosstalk between CD8+ T and NK cells: fine-tuning of antitumor immune response. [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 4059. doi:10.1158/1538-7445.AM2015-4059
Abstract 4080: Role of nuclear PKM2 in transcriptional regulation leading to EMT
Authors: Konno Masamitsu , Nishida Naohiro , Kawamoto Koichi , Koseki Jun , Doki Yuichiro , Mori Masaki , Ishii Hideshi
Published: August 2015

Pyruvate kinase M2 (PKM2) maintains aerobic glycolysis in cancer cells and stimulates cell proliferation by controlling Warburg effect(Nature, 2008). In addition, PKM2 translocates to nucleus in response to EGFR signaling and up-regulates Cyclin D1 expression by controlling its transcription (Nature, 2011). PKM2 confers malignant potential by controlling both metabolism and transcriptional regulation. In this study, we investigated the correlation between PKM2 and invasion and metastasis of colorectal cancer in terms of nuclear function, which has yet to be investigated. Tumor invasion is initiated with the change of epithelial-mesenchymal transition (EMT) in cancer cell, and we induced EMT by adding TGF-β1 and EGF to SW 480. After EGF induction, in parallel with E-cadherin down-regulation and Vimentin up-regulation, the expression of PKM2 was increased both in transcriptional and translational level and PKM2 translocated into nucleus. PKM2 knock-down by siRNA suppressed E-cadherin decrease and invasion capacity in invasion assay. We created sh pyruvate kinase SW 480 with mouse PKM1 or PKM2 was overexpressed both of which have DNA sequence resistant to sh pyruvate kinase. PKM2 overexpression stimulated EMT induction by enhancing E-cadherin decrease. TGIF2 was identified which interacted with PKM2 in nucleus in response to EMT induction, which was considered to have a key role in controlling EMT induction. We performed immunohistochemical staining with specific antibody to PKM2 using clinical samples of colorectal cancers. Clinicopathological analysis showed that PKM2 positivity significantly correlated with lymph node metastasis and distant organ metastasis. In conclusion, our data suggested that PKM2 nuclear function had a crucial role in controlling invasion and metastasis.

Citation Format: Masamitsu Konno, Naohiro Nishida, Koichi Kawamoto, Jun Koseki, Yuichiro Doki, Masaki Mori, Hideshi Ishii. Role of nuclear PKM2 in transcriptional regulation leading to EMT. [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 4080. doi:10.1158/1538-7445.AM2015-4080
Abstract 4082: PDLIM2 : A cytoskeleton to nuclear courier protein for the IGF-1, Wnt and TGF beta signalling pathways in Epithelial to Mesenchymal Transition
Authors: Bustamante Garrido Milan , Cox Orla T. , O'Flanagan Ciara , Buckley Deirdre A. , Kiely Patrick A. , O'Connor Rosemary
Published: August 2015

PDLIM2 is a PDZ-LIM domain protein that regulates the stability and activity of transcription factor families (including NFκB, STATs and beta catenin). PDLIM2 expression is repressed in certain cancers but it is also highly expressed in Triple Negative Basal Breast cancers that are characterized by poor survival. Suppression of PDLIM2 reverses the EMT phenotype, inhibits polarized cell migration, and disrupts formation of polarized epithelial acini in 3D cell cultures. PDLIM2 shuttles from the cytoskeleton to the nucleus, but what mediates this nuclear translocation or activity in transcription factor regulation is unknown.

The aim of this study was to identify the mechanisms governing PDLIM2 subcellular localization and nuclear translocation. We found that IGF-1or TGF-β promotes PDLIM2 accumulation in the nucleus. Similarly, WNT3a stimulation enhances PDLIM2 accumulation in the nucleus while inhibition of WNT activity results in PDLIM2 stabilization in the cytoplasm. Cytoplasmic to nuclear translocation is associated with reduced phosphorylation on several serine residues in PDLIM2. The de-phosphorylation and subsequent nuclear translocation of PDLIM2 can be prevented by inhibiting the protein phosphatase PP1. In contrast, PDLIM2 phosphorylation can be enhanced by activation of protein kinase C, which is dependent on the presence of the focal adhesion scaffolding protein RACK1 in a complex with PDLIM2.

Overall, the data indicate that PDLIM2 cytoplasmic to nuclear translocation in response to IGF-1, WNT or TGF beta signalling is mediated by serine phosphorylation and de-phosphorylation by cytoskeleton-associated kinases and phosphatases. Thus PDLIM2 acts as a “cytoskeleton to nucleus” courier protein for these signalling pathways to promote cancer cell migration and EMT.

Citation Format: Milan Bustamante Garrido, Orla T. Cox, Ciara O'Flanagan, Deirdre A. Buckley, Patrick A. Kiely, Rosemary O'Connor. PDLIM2 : A cytoskeleton to nuclear courier protein for the IGF-1, Wnt and TGF beta signalling pathways in Epithelial to Mesenchymal Transition. [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 4082. doi:10.1158/1538-7445.AM2015-4082
Abstract 4096: Identification of EMT inhibitors using novel zebrafish reporter lines
Authors: Jimenez Laura , Wang Jindong , Jette Cicely , Warner Steven L. , Bearss David , Stewart Rodney
Published: August 2015

The Epithelial to Mesenchymal Transition (EMT) plays a crucial role during development and aberrant reactivation of EMT in tumor cells promotes metastasis and drug resistance. Mechanisms that control EMT remain incompletely understood, hampering efforts to target EMT in cancer patients. To identify upstream inducers and downstream effectors of EMT we have developed a novel zebrafish neural crest (NC) EMT reporter line, Tg(snail1b:GFP), for in vivo screening of EMT and cell migration inhibitors. After screening a number of candidate kinase inhibitors for their ability to block NC migration in live Tg(snail1b:GFP) embryos, we found that the AXL receptor tyrosine kinase inhibitor, TP-0903 (HCI-2084), potently blocks hindbrain morphogenesis and cranial NC cell migration. Treatment of embryos at the 3-somite stage with TP-0903 significantly decreases twist1a expression, a key regulator of EMT, blocks differentiation of dlx2a-expressing chondrogenic precursors, and instead promotes the differentiation of mitfa-expressing melanophore precursors that form adjacent to the neural tube. RNA-Seq analysis revealed that TP-0903 treated embryos have a significant upregulation of retinoic acid (RA) signaling. Co-treatment of TP-0903 with DEAB, an RA inhibitor, rescues the hindbrain and NC migration and differentiation defects caused by TP-0903 treatment. We are currently investigating the potential role of AXL in NC cell migration and differentiation, and evaluating TP-0903 as a possible therapy for neuroblastoma, by using the zebrafish neuroblastoma pre-clinical model and human neuroblastoma cells in vitro.

Citation Format: Laura Jimenez, Jindong Wang, Cicely Jette, Steven L. Warner, David Bearss, Rodney Stewart. Identification of EMT inhibitors using novel zebrafish reporter lines. [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 4096. doi:10.1158/1538-7445.AM2015-4096
Abstract 4099: IGF1R signaling connects DNp73-mediated EMT with the acquisition of cancer stem cell properties
Authors: Meier Claudia , Steder Marc , Hardtstock Philip , Alla Vijay , Pützer Brigitte M.
Published: August 2015

Metastatic spread is the major cause of cancer-associated mortality and induced in primary tumor cells by the acquisition of mesenchymal features. Emerging evidence suggests that metastasis-initiating cancer cells undergoing epithelial-mesenchymal transition (EMT) possess stem cell-like traits. In this study, we demonstrate that oncogenic DNp73, an inhibitor of p53 tumor suppressor family members, has a fundamental role in driving both, transition into a mesenchymal state and providing cancer cells with stem cell-like properties through constitutive activation of IGF1R signaling. In patient melanoma tissues, DNp73 expression correlates with increased Breslow depth and is enriched in skin cancer metastases. Knockdown of endogenous DNp73 in highly metastatic cell lines abolishes the aggressive phenotype, whereas tumor xenografts overexpressing DNp73 possess significantly greater capacity to invade the surrounding tissue and form metastatic lesions. Transition from an epithelial to a motile mesenchymal phenotype is accompanied by a drastic reorganization of the cytoskeleton and modulation of cell-cell adhesion molecules. Importantly, regulation of EMT by DNp73 is induced through activation of IGF1R-AKT signaling followed by an increase of Slug and loss of E-cadherin. Additionally, we identified a novel interplay between DNp73 and IGF1R in the development of cancer stemness. Tumor spheres derived from metastatic melanoma cells with endogenously high DNp73 display a strong capacity for self-renewal and tumor growth after subcutaneous injection in mice compared to DNp73-depleted cancer stem cells. Our results clearly demonstrate that DNp73-driven stemness involves IGF1R activation. Selective inhibition of IGF1R blocks DNp73-mediated upregulation of Nanog, Oct4 and CD133, which is accompanied by a decrease in spheroid formation and tumor invasiveness. Taken together, our findings show for the first time that DNp73 is crucial to endow cancer cells with mesenchymal properties and the ability to self-renew, which is responsible for malignant progression and tumor dissemination. Moreover, we establish IGF1R as driving force for a DNp73-induced aggressive cancer phenotype, highlighting the DNp73-IGF1R cascade as promising therapeutic target to selectively prevent metastasis initiation.

Citation Format: Claudia Meier, Marc Steder, Philip Hardtstock, Vijay Alla, Brigitte M. Pützer. IGF1R signaling connects DNp73-mediated EMT with the acquisition of cancer stem cell properties. [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 4099. doi:10.1158/1538-7445.AM2015-4099
Abstract 4103: Knockdown of Notch1 inhibits tumor cell proliferation, migration, and invasion and reverses epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma
Authors: Guo Huajiao , Lu Yi , Qian Chaonan , Zhang Jian
Published: August 2015

Despite a large bodies of evidences support an oncogenic role of Notch signaling in several human solid cancers, however, the pleiotropic effects and molecular mechanisms of blockage of Notch signaling on nasopharyngeal carcinoma (NPC) remain unclear. In this study, Real-time RT-PCR and western blotting were firstly used to examine the expression of Notch1 receptor in a panel of NPC cell lines. CNE1 and CNE2 cells expressed a higher level of Notch1 compared to HONE1, SUNE1 and HK cells. Then Notch1 expression was specifically knocked down in CNE1 and CNE2 cells by Notch1 short hairpin RNA (shRNA). This resulted in a significant inhibition of cell proliferation, migration, and invasion; and interestingly, epithelial-mesenchymal transition (EMT) was reversed in these Notch1-transfected cells along with demonstration of epithelialioid-like morphology change and increase of E-cadherin and decrease of Vimentin expressions. In addition, targeted knockdown of Notch1 dramatically inhibited expression of urokinase plasminogen activator (uPA) and its receptor uPAR, chemokine CCL2 and CXCL16 indicating that these factors are downstream targets of Notch1. Finally, knockdown of Notch1 significantly diminished CNE1 cell growth in a murine model concomitant with inhibition of cell proliferation and induction of apoptosis. Taken together, these results suggest Notch1 may become a therapeutic target in nasopharyngeal carcinoma.

Note: This abstract was not presented at the meeting.

Citation Format: Huajiao Guo, Yi Lu, Chaonan Qian, Jian Zhang. Knockdown of Notch1 inhibits tumor cell proliferation, migration, and invasion and reverses epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma. [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 4103. doi:10.1158/1538-7445.AM2015-4103
Abstract 4110: Inhibition of angiopoietin 1/2 and c-MET impairs metastatic potential in a patient derived xenograft of renal carcinoma
Authors: Orillion Ashley , Adelaiye-Ogala Remi , Shen Li , Ciamporcero Eric , Ku Sheng Yu , Ramakrishnan Swathi , Elbanna May , Chintala Sreenivasulu , Pili Roberto
Published: August 2015

Background: Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and diagnoses have been on the rise since the 1990s. While patients with early stage ccRCC have more than a 50% survival rate over four years, those with advanced stage disease have a less than 10% chance of 5 year survival. In our current study, we utilized an angiopoietin 1/2 inhibitor (Amgen 386) and a novel c-MET inhibitor in murine models grafted with human derived xenografts (PDX) with a high metastatic penetrance. Our goal was to assess the ability of these compounds to prolong survival via metastatic inhibition. Methods: Seven week old SCID mice were implanted either subcutaneously or orthotopically with human ccRCC PDX RP-R-02LM. Approximately one month post implantation, mice were started on treatment with Angiopoietin 1/2 inhibitor (Amgen 386), c-met inhibitor (compound A) or a combination of the two. Amgen 386 was administered twice a week subcutaneously at 5.6mg/kg. Compound A was given daily via oral gavage at 30mg/kg. As a survival study, treatment was continued until the mice reached a point where they needed to be euthanized or they were found dead. Results: In our metastatic PDX model, RP-R-02LM, Amgen386 single agent treatment and combination c-MET inhibitor and Amgen386 treatment significantly reduced metastases significantly, p = 0.0075 and p = 0.0001 respectively. Further, an ongoing survival study has shown that at four and a half months post implantation both single agent and combination treatments prolong the survival of mice implanted with RP-R-02LM, a highly metastatic PDX. Conclusions: Our preliminary results suggest that Amgen386 in combination with a novel c-MET inhibitor are significantly inhibiting the spread of metastases and prolonging survival. This indicates a potential use for combination therapy in treating patients with ccRCC in advanced stages of the disease.

Citation Format: Ashley Orillion, Remi Adelaiye-Ogala, Li Shen, Eric Ciamporcero, Sheng Yu Ku, Swathi Ramakrishnan, May Elbanna, Sreenivasulu Chintala, Roberto Pili. Inhibition of angiopoietin 1/2 and c-MET impairs metastatic potential in a patient derived xenograft of renal carcinoma. [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 4110. doi:10.1158/1538-7445.AM2015-4110