bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 660: Modelling conventional and serrated colorectal tumorigenesis by altering the Wnt and MAPK signaling pathways
Authors: Whitehall Vicki , Fernando Winnie , McKeone Diane , Bettington Mark , Pearson Sally , Bond Catherine , Leggett Barbara
Published: July 2016

Background. The majority of conventional colorectal adenomas are initiated by activation of the Wnt signal, whilst activation of the MAPK pathway is associated with progression to cancer. In contrast, sessile serrated adenomas are initiated by activation of the MAPK signaling pathway due to mutation of the BRAF oncogene, whilst the Wnt pathway is activated at the transition to dysplasia. We hypothesised that whilst both the Wnt and MAPK pathways play key roles in colorectal tumorigenesis, the order in which these signaling pathways are activated would determine the morphology of the lesion arising.

Methods. To test this hypothesis, we utilised a conditional BRAF mutant mouse model crossed with an inducible Cre driven by the murine Villin gene promoter, to restrict Cre expression and induction of the BRAF mutation to the intestine. The MAPK pathway was activated two weeks after birth, whilst activation of the Wnt signal was achieved using APCMin mice or the intestinal carcinogen azoxymethane. Morphology and number of lesions was confirmed by histologically examination. Nuclear β-catenin immunostaining provided a surrogate for activation of the Wnt signal.

Results. Induction of the BRAF mutation resulted in the rapid and sustained development of widespread intestinal hyperplasia. Murine serrated adenomas (mSA) mimicking the morphology of human traditional serrated adenomas were observed by 5 months and by 10 months 8/18 (44.4%) mice had developed mSA, primarily in the duodenum. Activation of the Wnt pathway using azoxymethane dramatically accelerated the serrated phenotype in both the small intestine (2.9 mSA per mouse, P<0.01) and colon (3.1 mSA per mouse, P<0.05) by 3 months. The MAPK and Wnt pathways also synergized in APCMin mice, increasing the average number of conventional adenomas per mouse from 2.1 to 78.8 (P<0.001).

Conclusion. The rapid induction of hyperplasia and exclusive development of serrated lesions demonstrates that BRAF mutation is a key molecular event underlying initiation of serrated neoplasia. Importantly, we have demonstrated that activation of the MAPK pathway through BRAF mutation is sufficient for commitment to the serrated pathway and that whilst subsequent activation of the Wnt signal can accelerate tumorigenesis, it is not sufficient to override the existing serrated morphology.

Citation Format: Vicki Whitehall, Winnie Fernando, Diane McKeone, Mark Bettington, Sally Pearson, Catherine Bond, Barbara Leggett. Modelling conventional and serrated colorectal tumorigenesis by altering the Wnt and MAPK signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 660.
Abstract 664: Insulin receptor signaling contributes to mammary tumorigenesis in mice
Authors: Poloz Yekaterina , Mouaaz Samar , Lam Sonya , Stambolic Vuk
Published: July 2016

A recent convergence of clinical and epidemiological evidence suggests that hyperinsulinemia associated with obesity and type 2 diabetes leads to higher incidence and poor outcome of breast cancer (BC). To probe the role of insulin signaling in BC, we created the MMTV-driven polyoma middle T BC model mice with an inducible mammary epithelium-specific deletion of the insulin receptor (INSR). Deletion of INSR in the mammary gland considerably reduced the mammary tumour burden in the INSR knockout mice as compared to the INSR wildtype controls, without affecting the tumour onset. The average weight of individual tumours was similar across the genotypes, suggesting that deletion of INSR affected the tumour initiation potential of the epithelial cells rather than tumour growth. Deletion of INSR also affected the metastasis of mammary tumours to the lung, with the INSR knockout mice presenting with half the number of metastases compared to the INSR wildtype controls. Our ongoing efforts are focused on mapping the signaling events downstream of the INSR that govern tumour initiation and metastasis in our mice, as well as modeling the impact of obesity on tumors in this model system, with the ultimate goal of identifying the role of INSR signaling in BC development and progression.

Citation Format: Yekaterina Poloz, Samar Mouaaz, Sonya Lam, Vuk Stambolic. Insulin receptor signaling contributes to mammary tumorigenesis in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 664.
Abstract 665: Blocking IL-1-NF-kB axis inhibits Kras-induced pancreatic ductal adenocarcinoma
Authors: Ling Jianhua , Fu Jie , Wu Min , Cao Yu , Chiao Paul
Published: July 2016

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer mortality in the USA with approximately 50 000 new cases and 40 000 deaths in 2015.The 5-year survival rate has remained at 5% for the past three decades and the median survival duration is less than 6 months. The very high rate of mutant Kras occurs in pancreatic cancer (>90%) and pancreatic intraepithelial neoplasia (panIN) (>50%). However, the key signaling pathway of downstream Kras remains unidentified.

We previously uncovered that the NF-êB signaling pathway was constitutively activated in most PDAC specimens and pancreatic cancer cell lines. More recently, our results showed that constitutive NF-êB activity in PDAC was induce by IL-1 autocrine mechanism. IL-1/p62 feedforward loops that initiated by the mutant Kras sustained NF-êB activity and were required for pancreatic cancer development. Therefore, we hypothesize that blocking IL-1 - NF-êB signaling pathway will inhibit Kras-induced pancreatic ductal adenocarcinoma.

To identify the mechanistic role of mutant Kras-induced IL-1 expression in PDAC, we generated genetically engineered mice of Pdx1-cre/Kras LSL-G12D/p53 M/+IL-1á−/−. Our findings revealed that these mutant mice had developed significantly less PDAC number and a significantly delayed PDAC at old ages. Furthermore, we assess the effect of pharmacological IL-1R1 antagonist (anakinra) in human pancreatic cancer cell lines and orthotopic pancreatic cancer mouse. We found that IL-1R1 antagonist significantly decreased NF-êB activity and inhibited cell proliferation, migration and invasion. More interestingly, the combinational treatment IL-1R1 antagonist plus gemcitabine significantly reduced the tumor burden in orthotopic pancreatic cancer mouse.

These results suggested that pharmacologically targeting IL-1á may provide a novel therapeutic approach for pancreatic cacner.

Citation Format: Jianhua Ling, Jie Fu, Min Wu, Yu Cao, Paul Chiao. Blocking IL-1-NF-kB axis inhibits Kras-induced pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 665.
Abstract 677: EML4-ALK fusion gene expressing transgenic mouse for lung cancer model
Authors: Pyo Kyoung-Ho , Kim Hye Ryun , Lim Sun Min , Yun Mi Ran , Kim Sung-Moo , Kim Hwan , Kang Han Na , Lee Ji Min , Kim Sang Gyun , Cho Byoung Chul
Published: July 2016

Background: EML4-ALK is a fusion-type protein typrosin kinase that is generated in lung cancer. We have established transgenic mouse line. The surfactant protein C gene (SPC) is only expressed in alveolar epithelial cells. We applied SPC promoter on encoded human-EML4-ALK gene. Normally, EML4-ALK gene is silenced when LoxP-PGK-LoxP gene is turned on. When Cre recombinase is present with the specific inducer, tamoxifen, EML4-ALK gene is expressed in alveolar epithelial cell, and converses to lung cancer.

Materials and Methods: For generation of EML4-ALK fusion transgenic mouse, a cDNA fragment encoding FLAG tagged EML4-ALK (variant 1) was ligated to end of the CMV-LoxP-PGK-LoxP. The expression cassette was injected into pronuclear-stage embryos of B6 mouse to generate Tg mouse. Transgenic mouse was prepared for interbreed with KI mouse (SPC-CreER2T-trTA-NeoR). The KI-Tg fusion mouse was confirmed by genotyping, then the mouse was injected with tamoxifen (cre recombinase inducer) intraperitoneally. The lung tumors were observed by MRI or CT. The lung pathology was analyzed by immunohistochemistry and hematoxylin & eosin staining. For confirmation of EML4-ALK protein expression, western blot was performed in different organs. EML4-ALK tumor-bearing mouse was treated with ALK tyrosine kinase inhibitor (TKI) for 2 weeks and tumor shrinkage was measured.

Results: EML4-ALK tumor was observed after one week. Tumor nodules were widely observed in all lung lobes. Pathologically, the histologic type was adenocarcinoma and had homology to patient tissue on H&E stain. We confirmed EML4-ALK expression in different organs including lung, heart, liver, stomach, kidney, colon and brain. EML4-ALK (120kDa) was strongly expressed in lung, but not

in other organs. Indeed, ALK and Flag expression were observed in tumor region by immunohistochemistry. The EML4-ALK tumor bearing mice were treated with ALK TKI for 2 weeks. The ALK TKI-treated mice showed dramatic shrinkage of tumors and showed gain in body weight.

Conclusions: EML4-ALK Tg mouse was successfully established. The benefits of the model compared to a previous conditioned Tg mouse model are (1) no need to treat Cre inducer daily, and (2) short duration (approximately 1 week) of tumorigenesis. We confirmed dramatic tumor shrinkage after treatment with ALK TKI. This model provides a strong preclinical model for testing ALK TKIs in lung cancer.

Citation Format: Kyoung-Ho Pyo, Hye Ryun Kim, Sun Min Lim, Mi Ran Yun, Sung-Moo Kim, Hwan Kim, Han Na Kang, Ji Min Lee, Sang Gyun Kim, Byoung Chul Cho. EML4-ALK fusion gene expressing transgenic mouse for lung cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 677.
Abstract 680: Diet-induced obesity promotes tumor aggressiveness in genetically engineered mouse models of endometrial hyperplasia/cancer
Authors: Clark Leslie H. , Zhou Chunxiao , Montgomery Stephanie A. , Gehrig Paola A. , Bae-Jump Victoria
Published: July 2016

Objective: To determine the effect of diet-induced obesity on the development of endometrial hyperplasia and cancer in genetically engineered mouse models.

Methods: The PTEN heterozygous mouse model for endometrial hyperplasia and the LKB1/p53 endometrioid endometrial cancer mouse model were used. Mice were divided into two dietary groups and fed either a high-fat diet (HFD, 60% calories from fat, obese) or a low-fat diet (LFD, 10% calories from fat, lean) beginning at 3 weeks of age until sacrifice at 32 weeks (PTEN model) and 12 weeks (LKB1/p53 model). At the time of sacrifice, mouse weight and uterine/tumor size were documented. A dedicated mouse pathologist reviewed uterine pathology. For the PTEN heterozygous mouse model, uterine pathology was determined to be simple hyperplasia (SH) if increased number of simple tubular glands were seen without atypia. Complex hyperplasia (CH) was defined as branching, papillary lumenal infoldings, or back-to-back glandular crowding with or without nuclear atypic.

Results: A total of 30 PTEN mice were examined, 14 in the obese group and 16 in the lean group. The HFD-fed mice were larger than the LFD-fed mice with a mean weight of 38.1 gm compared to 29.7 gm (p<0.001). There was no difference in uterine weight between the obese and lean mice, 0.30 versus 0.34 gm, respectively (p = 0.76). The lean mice were more likely to develop SH, 44% (n = 7) versus 7% (n = 1) in the obese group (p = 0.04). Of the obese mice, 57% (n = 8) developed CH (± atypia) compared to 31% (n = 5) of the lean mice (p = 0.16). A total of 35 LKB1/p53 mice were examined with 21 in the obese group and 14 in the lean group. The obese LKB1/p53 mice were larger (32.8 gm) than the lean mice (24.0 gm) (p = 0.033). The majority of the LKB1/p53 mice developed endometrioid adenocarcinoma with increased tumor size in the obese group (2.1 gm) versus lean group (0.79 gm) (p = 0.04).

Conclusions: Diet-induced obesity led to increased mean body weight in both mouse models. In the PTEN heterozygous mouse model, a trend towards more complex endometrial hyperplasia was found in the obese group along with a higher rate of simple hyperplasia in the lean group. Increased endometrial tumor size was seen in the obese versus lean LKB1/p53 mice, suggesting that obesity may lead to increased aggressiveness in endometrial cancer.

Citation Format: Leslie H. Clark, Chunxiao Zhou, Stephanie A. Montgomery, Paola A. Gehrig, Victoria Bae-Jump. Diet-induced obesity promotes tumor aggressiveness in genetically engineered mouse models of endometrial hyperplasia/cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 680.
Abstract 685: Selective inhibitor of nuclear export (SINE) compounds prevent migration and proliferation of triple negative breast cancer (TNBC) cells by restoring expression of ARRDC3
Authors: Soung Young Hwa , Kashyap Trinayan , Nguyen Thalia , Landesman Yosef , Chung Jun
Published: July 2016

Arrestin-related domain-containing protein-3 (ARRDC3) is one of 6 mammalian arrestins, which has been shown to possess tumor suppressor activity by inducing degradation of phosphorylated â2-adrenergic receptor (â2 AR) and integrin â4 (ITG â4). Our previous studies demonstrated that Sirt2 epigenetically silenced expression of ARRDC3 in TNBC cells and overexpression of ARRDC3 significantly reduced their invasive potential. Therefore, we hypothesized that compounds, which restore ARRDC3 expression levels could serve as novel therapeutic options for TNBC. To test this, we examined the ability of SINE compounds: KPT-185 and KPT-330 (selinexor), to effectively restore ARRDC3 expression. We observed that KPT-185 and selinexor significantly induced ARRDC3 expression and inhibited the proliferation and the invasiveness of the TNBC cell lines MDA-MB-231 and MDA-MB-468 in vitro. In vivo, selinexor inhibited the tumor growth of MDA-MB-231 and MDA-MB-468 xenografts by nearly 100% compared with vehicle treated animals. Furthermore, immunohistochemical analysis of TNBC tumors from selinexor treated mice revealed increased ARRDC3 expression versus vehicle treated animals. Our results suggest that restoration of ARRDC3 expression is an important antineoplastic mechanism of SINE compounds in TNBC and suggest that selinexor could be an effective treatment against tumors with downregulated ARRDC3.

Citation Format: Young Hwa Soung, Trinayan Kashyap, Thalia Nguyen, Yosef Landesman, Jun Chung. Selective inhibitor of nuclear export (SINE) compounds prevent migration and proliferation of triple negative breast cancer (TNBC) cells by restoring expression of ARRDC3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 685.
Abstract 692: Identifying genes that regulate bladder cancer progression and invasion
Authors: Han Amy L. , Veeneman Brendan , Tomlins Scott A. , Keller Evan T.
Published: July 2016

Transitional cell carcinoma (TCC) is the most common type of bladder cancer and can be categorized as either non-muscle invasive (Ta-T1) or muscle invasive (≥T2). Approximately 50% of bladder cancers are T1 at initial diagnosis; however, the recurrence rate for these tumors is high and they may progress into T2. In this study, we aimed to determine if there are specific gene expression differences between T1 vs. T2 bladder cancer that can help identify key regulators in bladder cancer progression and invasion. T1 and T2 bladder cancer tissues were subjected to RNA-Seq to evaluate for differences among these stages. Additionally, the Oncomine database was examined to further narrow down potential candidates that differentiate T1 from T2. These efforts led to the identification of an extracellular matrix glycoprotein, fibulin-3 (FBLN3), as being highly expressed in T2 compared to T1 tissues. To validate these findings, FBLN3 expression was measured using qRT-PCR from formalin fixed and paraffin embedded tissues from patient bladder samples ranging from stages Ta-T4. These studies confirmed that FBLN3 expression was elevated in muscle-invasive compared to non-muscle invasive bladder cancer. Consistent with these findings, FBLN3 expression level correlated with the invasive ability of several bladder cancer cell lines. Specifically, FBLN3 expression was determined using both qRT-PCR and western blotting amongst the T24, UMUC-13, UMUC-3, RT4, and 5637 bladder cancer cell lines. The most invasive cell lines, T24 and UMUC-13, demonstrated the highest FBLN3 expression. In contrast, the least invasive cells, RT4 and 5637, demonstrated the least FBLN3 expression. To determine a functional role for FBLN3 in bladder cancer invasion, we knocked down or increased FBLN3 expression in bladder cancer cell lines using lentiviral transduction. Knockdown of FBLN3 expression in the T24 and UMUC-13 cells inhibited the invasion and migration of these bladder cancer cells; whereas, FBLN3 overexpression in the 5637 cells promoted the invasiveness of the bladder cancer cells. Furthermore, cell viability and growth rates were not affected by manipulation of FBLN3 expression. Our results indicate that FBLN3 serves as a pro-invasive factor in bladder cancer. These findings suggest that FBLN3 could serve as (1) a biomarker to differentiate T1 from T2 bladder cancers and (2) a promising therapeutic target.

Citation Format: Amy L. Han, Brendan Veeneman, Scott A. Tomlins, Evan T. Keller. Identifying genes that regulate bladder cancer progression and invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 692.
Abstract 699: Inhibition of cathepsin L1 by U87 KIF9 suppression in glioblastoma multiforme: a potential therapeutic target
Authors: Rubinstein Maria M. , Segall Jeffrey
Published: July 2016

Glioblastoma multiforme is a fatal primary brain cancer. It has remained one of the most difficult cancers to treat because of glioblastomas’ aggressive and local invasive nature. Glioblastoma tumors have many myeloid cells, especially microglia. Increased levels of microglia result in increased glioblastoma invasion and are associated with worse clinical outcomes. Glioblastomas’ infiltrative mechanisms are mostly unknown which hinder the efficacy of current treatments. Therefore, further investigations on the mechanisms responsible for local invasion are potential targets for therapeutic interventions.

Recently, there has been interest in evaluating the role of kinesin family members (KIFs) and cathepsins (CTS) as possible targets for therapeutic interventions. KIFs play an essential role in both physiological axonal transport processes and in processes of cell division. Previous studies have investigated the inhibition of KIF11 as a possible therapeutic target in glioblastoma. Preliminary studies in our group have found that the inhibition of KIF9 reduces microglia-stimulated glioma invasion. CTS, which are proteases that function in many pathways and increase neoplastic progression, are other possible therapeutic targets. Specifically in glioblastoma, inhibition of cathepsin L1 (CTSL1) (i) promotes tumor cell death, (ii) decreases apoptosis threshold and (iii) sensitizes glioblastoma cells to radiation. Our study investigated the role of KIF9 suppression in secretion and production of CTSL1.

In order to investigate this relationship, U87 cells were incubated with and without the presence of microglia. Non-targeting and stable KIF9 knockdown cells were incubated with and without the presence of microglia. CTSL1 concentration levels were measured by enzyme immunoassay (ELISA). We investigated CTSL1 concentrations in the cell lysates as well as the cell supernatants. Our results demonstrated an overall decrease in production and secretion of CTSL1 in KIF9 knockdown cells as compared to non-targeting shRNA cells. The effect of KIF9 knockdown was evident in the presence and absence of microglia. Although the exact mechanism needs to be further clarified, inhibition of CTSL1 by KIF9 suppression could be a possible potential target for therapeutic intervention.

Citation Format: Maria M. Rubinstein, Jeffrey Segall. Inhibition of cathepsin L1 by U87 KIF9 suppression in glioblastoma multiforme: a potential therapeutic target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 699.
Abstract 702: Serglycin promotes cell migration via modulating cytoskeleton reorganization and enhancing focal adhesion turnover
Authors: Chen Jeou-Yuan , Guo Jing-You
Published: July 2016

Serglycin (SRGN), a hematopoietic cell granule proteoglycan, has recently been shown to be overexpressed in several aggressive cancer types. In nasopharyngeal and hepatocellular carcinomas, elevated expression of SRGN was shown to correlate with poor prognosis. However, the molecular mechanisms underlying SRGN-mediated malignancies remain to be explored. In this study, we showed that SRGN is expressed at elevated levels in several lung cancer-derived cell lines. By gain-of-function and loss-of-function approaches, we showed that SRGN promoted NSCLC cell migration. SRGN modulated actin cytoskeleton reorganization and promoted lamellipodia and filopodia formation at the leading edge, facilitating a directional movement during wound closure in NSCLC cells. In consistence, increased levels of activated Rac1, which is required for lamellipodia formation, and CDC42, which is required for filopodia formation, were detected. Increased focal adhesion (FA) turnover, the process of continuous assembly and disassembly of adhesions, would lead to increased cell migration. We further showed that SRGN promoted Src activation, leading to increased phosphorylation of paxillin (PAX) at Y118 as well as reduced PAX/FAK complex formation, which were well-defined indicators of FA turnover. Taken together, these data suggested that SRGN promotes cell migration through inducing cytoskeleton reorganization and focal adhesion turnover.

Citation Format: Jeou-Yuan Chen, Jing-You Guo. Serglycin promotes cell migration via modulating cytoskeleton reorganization and enhancing focal adhesion turnover. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 702.
Abstract 705: PRL-3/PTP4A3 phosphatase promotes uveal melanoma cell migration through the regulation of integrin â1 in focal adhesion
Authors: Foy Malika , Anezo Oceane , Planque Nathalie , Saule Simon
Published: July 2016

Using transcriptomic analysis, we found that Phosphatase of Regenerating Liver-3 (PRL-3) overexpression is highly correlated with metastatic tumors and predicts poor prognosis in patients with uveal melanoma. PRL-3 is a dual specific phosphatase with a carboxy-terminal farnesylation motif, allowing anchorage to the plasma membrane. PRL-3 overexpression in ocular melanoma cell line-1 (OCM1) significantly increases cell migration in vitro and invasiveness in vivo, suggesting a direct role of PRL-3 in metastasis (Laurent et al, Cancer Res 2011; 71:666). Despite its role in the metastatic process of various cancers, its mechanism of action and its intracellular substrates remain largely unknown. It has been shown that the anchorage of PRL-3 to the plasma membrane plays an important role in the metastatic process, suggesting that the phosphatase regulates transmembrane substrates. Among potential targets involved in extracellular matrix adhesion, we focused on integrin â1 which we found overexpressed in the transcriptomic analysis of our uveal melanoma cohort.We showed that FTI-277, a farnesyltransferase inhibitor that prevents PRL-3 anchorage to the plasma membrane, abolishes PRL-3-induced OCM1 cell migration on collagen I in vitro, specifically enhances the spreading of OCM1 cells overexpressing PRL-3, and allows the formation of large focal adhesions involving integrin â1. Knockdown of integrin â1 in OCM1-PRL3 cells partially restores the spreading and migration. Furthermore, in focal adhesions, we observed that PRL-3 specifically regulates the aggregation of integrin â1 but does not affect integrin â3. Our results suggest the involvement of integrin â1 in PRL-3-mediated cell migration. To go further, we are currently investigating the phosphorylation status of specific integrin â1 residues, known to be involved in cell adhesion. The present study highlights the importance of PRL-3 membrane localization in uveal melanoma cell migration, by regulating adhesion to the extracellular matrix through integrin â1.

Citation Format: Malika Foy, Oceane Anezo, Nathalie Planque, Simon Saule. PRL-3/PTP4A3 phosphatase promotes uveal melanoma cell migration through the regulation of integrin â1 in focal adhesion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 705.
Abstract 708: Defensin-alpha 6 (DEFA6) as a protumorigenic function in vitro and prognostic marker in colorectal cancer
Authors: Jeong Dongjun , Kim Hyungjoo , Oh Seunghyun , Ban Seona , Ji Sanghee , Kim Han Jo , Ahn Tae Sung , Kim Tae Hyun , Kwon Hyog Young , Jeon Seob , Bae Sang Byung , Kim Chang-Jin , Lee Moon Soo , Baek Moo-Jun
Published: July 2016

Objective

DEFA6 also known as human alpha defensing 6 (HD6) is a member of the alpha defensins family that defends the host against bacteria and viruses. Expression of DEFA6 is enhanced in colorectal cancer tissue and cells. The mechanism and function of DEFA6 have not been reported to play an important role in carcinogenesis and cancer progression. The goal of this study was to evaluate the protumorigenic functions of DEFA6 in the colorectal cancer cell line and to evaluate the clinical significance of DEFA6 expression in colorectal cancer patients.

Methods

DEFA6 was highly expressed in colorectal cancer cell lines. Colorectal cancer cell, HCT116, expressing high level of DEFA6 was used for the biological roles of DEFA6 by siRNA transfection. The protumorigenic functions of DEFA6 was evaluated by MTT assay, migration and invasion transwell assay and plate colony forming assay. DEFA6 expression was investigated by immunohistochemistry in 60 cases of colorectal cancer tissue and the association of DEFA6 expression was correlated with patient's survival.

Results

Immunohistochemistry analysis showed that the DEFA6 protein was expressed higher in death group (survival <5 years) than survival group (survival >5 years), (p = 0.02). The HCT116 of knockdowned DEFA6 by siRNA showed significant decrease in proliferation by MTT assay (p<0.05) and plate colony forming assay (p = 0.00). Also, the ability of migration and invasion was significantly reduced in DEFA6 knockdowned HCT116 compared to control HCT116 cell (both, p<0.05).

Conclusion

DEFA6 plays important roles in invasion and migration of colorectal cancer cell according to the functional study. As patient's survival mostly depends on the migration and invasion of the tumor cells, the high expression of DEFA6 in colorectal cancer is associated with patient's survival and could be a good prognostic marker.

Citation Format: Dongjun Jeong, Hyungjoo Kim, Seunghyun Oh, Seona Ban, Sanghee Ji, Han Jo Kim, Tae Sung Ahn, Tae Hyun Kim, Hyog Young Kwon, Seob Jeon, Sang Byung Bae, Chang-Jin Kim, Moon Soo Lee, Moo-Jun Baek. Defensin-alpha 6 (DEFA6) as a protumorigenic function in vitro and prognostic marker in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 708.
Abstract 709: In vitro functional study of novel oncogene serine protease 33 (PRSS33) and the clinical significance of PRSS33 expression in colorectal cancer patients
Authors: Jeong Dongjun , Ban Seona , Kim Hyungjoo , Oh Seunghyun , Ji Sanghee , Kim Han Jo , Ahn Tae Sung , Kim Tae Hyun , Kwon Hyog Young , Jeon Seob , Bae Sang Byung , Kim Chang-Jin , Lee Moon Soo , Baek Moo-Jun
Published: July 2016

Background: PRSS33, one of serine protease multigene family, has central roles in the regulation of a wide variety of physiological processes, including inflammation, development and malignancy. However, the function of this gene in colorectal cancer has not been elucidated. The goal of this study was to evaluate the oncogenic functions of PRSS33 in the colorectal cancer cell line and to evaluate the clinical significance of PRSS33 expression in colorectal cancer patients.

Method: PRSS33 was highly expressed in colorectal cancer cell lines, HCT116, SW480 and SW620. The oncogenic functions were evaluated in the cell lines by knocking down PRSS33 with siRNA transfection and compared them with PRSS33 highly expressing control cell lines. The functional studies included cell proliferation assay, invasion assay, migration assay and anchorage-independent semisolid agar colony forming assay. The clinical significance of PRSS33 expression was evaluated in 92 cases of colorectal cancer tissue by immunohistochemistry.

Results: The PRSS33 knockdowned cell lines by siRNA transfection showed significant decreases of proliferation, invasion, migration compared to those of control (p<0.05) respectively. The oncogenic function of PRSS33 was confirmed by anchorage-independent semi-solid agar colony forming assay. The PRSS33 knockdowned cell lines revealed lower colony formation on semisolid agar compared to the PRSS33 highly expressing control cell lines. The disease-free survival rate was decreased in patients of PRSS33 high expression (p = 0.001). The overexpression of PRSS33 was associated with survival and death by chi-square test (p = 0.002). Multivariate Cox-regression analysis showed an association between PRSS33 expression and prognosis (HR = 2.71, 95% CI = 1.39-5.27: p = 0.003).

Conclusion: This study indicates that PRSS33 is a novel pro-oncogene and the expression is an independent prognostic factor in colorectal cancer patients. In the future, research on the oncogenic signal pathway of PRSS33 in colorectal cancer is necessary.

Citation Format: Dongjun Jeong, Seona Ban, Hyungjoo Kim, Seunghyun Oh, Sanghee Ji, Han Jo Kim, Tae Sung Ahn, Tae Hyun Kim, Hyog Young Kwon, Seob Jeon, Sang Byung Bae, Chang-Jin Kim, Moon Soo Lee, Moo-Jun Baek. In vitro functional study of novel oncogene serine protease 33 (PRSS33) and the clinical significance of PRSS33 expression in colorectal cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 709.
Abstract 710: Epidermal growth factor-induced pyruvate dehydrogenase kinase 1 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation
Authors: Chen Ben-Kuen , Hsu Jinn-Yuan , Chang Wen-Chang
Published: July 2016

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of pyruvate dehydrogenase kinase 1 (PDK1) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced PDK1 expression in HNSCC. The tumor cell transformation induced by EGF was repressed by the depletion of PDK1. The down-regulation of PDK1 expression or inhibition of its activity significantly blocked EGF-enhanced cell migration and invasion. In addition, depletion of PDK1 impeded EGF-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastastic seeding of the lungs. Knockdown of PDK1 also inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. These results demonstrate that EGF-induced PDK1 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of PDK1 may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.

Citation Format: Ben-Kuen Chen, Jinn-Yuan Hsu, Wen-Chang Chang. Epidermal growth factor-induced pyruvate dehydrogenase kinase 1 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 710.
Abstract 711: The essential role of NKX6.3 in gastric cancer cell migration and invasion through targeting of Wnt/â-catenin and Rho-GTPase signaling pathways
Authors: Yoon Jung Hwan , Choi Won Suk , Park Won Sang
Published: July 2016

Despite ongoing research and recent progress, the prognosis for patients with advanced gastric cancer remains poor. Wnt/â-catenin and Rho-GTPase signaling pathways are known to play essential roles in malignant transformation and progression of various tumours, including gastric cancer. Here, we identify that NKX6 transcription factor, locus 3 (NKX6.3) binds directly to specific promoter regions of Wnt/â-catenin and Rho-GTPase pathway-related genes, resulting in inhibition of cancer cell migration and invasion. Additionally, we find that the expression level of NKX6.3 is involved in regulation of gastric cancer progression and expression of Wnt/â-catenin and Rho-GTPase pathway-related genes in clinical samples. These results suggest that NKX6.3 prevents EMT and cell migration, implying that NKX6.3 inactivation might be one of the key mechanisms of gastric cancer cell invasion and metastasis and might serve as a potential candidate for targeted gastric cancer therapy.

Citation Format: Jung Hwan Yoon, Won Suk Choi, Won Sang Park. The essential role of NKX6.3 in gastric cancer cell migration and invasion through targeting of Wnt/â-catenin and Rho-GTPase signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 711.
Abstract 712: Mechanisms of stromal Lkb1 loss induced tumorigenesis in mouse models of Peutz-Jeghers syndrome
Authors: Ollila Saara , Vaahtomeri Kari , Wong Iris , Laajanen Kaisa , Mäkelä. Tomi P.
Published: July 2016

Germline mutations in tumor suppressor kinase Lkb1 predispose to Peutz-Jeghers syndrome (PJS), with highly penetrant gastrointestinal polyposis and increased cancer risk. PJS polyps display abnormal growth of both stromal and epithelial cells. We have identified clonally expanding fibroblasts as the drivers of tumorigenesis in PJS mouse models by using Fsp1-Cre and Twist2-Cre mice to restrict Lkb1 deletion to stromal cells (unpublished), highlighting the importance of stromal-epithelial signaling in PJS tumorigenesis.

Here, we address the molecular mechanisms of the pathogenesis linked to stromal Lkb1 loss in PJS mouse models. Lkb1 is involved in tissue size control by inhibiting of mTORC1 pathway via AMPK (Shaw et al, Cancer Cell 2004) and by regulating Hippo pathway (Mohseni et al, Nat Cell Biol 2014). First, we investigated the involvement of Lkb1-AMPK-mTORC1 pathway in polyposis. We conditionally deleted both catalytic AMPKá subunits (á1 and á2) in mice using Fsp1-Cre. Surprisingly, Fsp1-Cre-AMPK mice did not develop any tumors by 17 months of age indicating that AMPK is not the critical mediator of Lkb1 tumor suppression in PJS. We next studied the Hippo pathway in polyps of PJS mouse models. Levels of Yap and Taz, the main downstream effectors of Hippo, were elevated in polyps as shown by Western blotting. Immunohistochemical staining revealed profound nuclear Yap/Taz localization indicating transcriptional activity in the stromal compartment throughout the polyps. In contrast, nuclear Yap/Taz staining in epithelial cells was only noted in a restricted polyp base stem cell zone. In addition, we performed RNA-seq analysis of the of Fsp1-Cre;Lkb1flox mouse polyps and noted shared gene expression changes with PJS patient polyps. RNA-seq also revealed significant enrichment of Yap signature, which was validated by qPCR. Finally, we used small intestinal epithelial organoid culture to study the potential of Yap/Taz induced secreted factors to stimulate growth of epithelial cells. We observed that Wnt5a and Epiregulin enhanced epithelial organoid growth, indicating them as candidates mediating the stromal-epithelial signaling and promoting tumorigenesis in PJS polyps.

In conclusion, we show that stromal Lkb1 mutations lead to phenotypes identical to germline Lkb1 heterozygosity in mice and propose increased stromal Yap/Taz activity as a potential mechanism to drive PJS tumorigenes.

Citation Format: Saara Ollila, Kari Vaahtomeri, Iris Wong, Kaisa Laajanen, Tomi P. Mäkelä. Mechanisms of stromal Lkb1 loss induced tumorigenesis in mouse models of Peutz-Jeghers syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 712.
Abstract 723: Distinguishing macrophages by a unique gene signature to measure response to treatment
Authors: Lyons Yasmin A. , Pradeep Sunila , Hansen Jean M. , Previs Rebecca A. , Yao Hui , Baggerly Keith A. , Sood Anil K.
Published: July 2016

Objectives: Tumor-associated macrophage infiltration is associated with poor prognosis, making macrophage depletion an ideal therapy. This study aims to identify a macrophage-specific gene signature to quantify macrophage content and measure response to treatment of high-grade serous cancer (HGSC) with colony stimulating factor-1 receptor (CSF1R) inhibitor.

Methods: 39 murine genes were identified in the literature as being macrophage-specific. Corresponding human orthologous genes were identified using BioMart database. Expression levels (log-transformed normalized RSEM values) of human homologous genes were retrieved from The Cancer Genome Atlas (TCGA) portal. Principal component (PC) and hierarchical clustering (HC) analysis of 305 primary HGSC was performed. Q-PCR was performed on human macrophage cell line, endothelial cell line, and fibroblast cell line. Q-PCR was also performed on tumor tissue obtained from CD57Bl/6 mice injected with murine ovarian cancer and treated with CSF1R inhibitor.

Results: 37 human homologous genes were identified for the macrophage specific murine genes. Of the 37 genes, 12 tracked together tightly and included the top 3 genes with greatest median absolute deviance of RSEM values, thus were chosen for further analysis. Q-PCR using human macrophage cell line revealed high expression in all 12 genes (delta CT <25), with TBXAS, CD14, and FCGR1A being the highest. All 12 genes showed greater expression in macrophages versus fibroblasts and endothelial cells, to varying degrees. Expression of CD14, TLR7, FCGR2C, and FCGR1B was significantly higher (p<0.0001) in macrophages compared to both fibroblasts and endothelial cells. Of these 12 human genes, 4 had homologous murine genes. These genes were used for Q-PCR on murine tumor tissue treated with CSF1R inhibitor. There was a statistically significant decrease in the level of CSF3R (p = 0.005), FCGR (p = 0.02), and PON3 (p = 0.002) between the control group and the group treated with CSF1R inhibitor.

Conclusions: These data suggest that macrophage specific genes could be useful for quantifying macrophage content in tumor samples to monitor therapy-related changes.

Citation Format: Yasmin A. Lyons, Sunila Pradeep, Jean M. Hansen, Rebecca A. Previs, Hui Yao, Keith A. Baggerly, Anil K. Sood. Distinguishing macrophages by a unique gene signature to measure response to treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 723.
Abstract 724: MicroRNA-100/mTOR/IL-1ra signaling maintains TAMs phenotype and enhances tumor cell stemness property in mouse breast cancer
Authors: Wang Wei , Liu Yan , Guo Jian , He Huiwen , Xie Junling , Chen Chong , Luo Yunping
Published: July 2016

Tumor-associated macrophages(TAMs),the main part of immune cells in tumor microenvironment(TME),play a potent role in promoting tumorigenesis. MicroRNAs (miRs) are considered to be crucial regulators in tumor progression. However, the role that miRs play in TAMs phenotype regulation still remains unclear.

In this study, we demonstrated that miR-100 played an important role in maintaining TAMs phenotype and enhancing tumor stemness properties. First, miRNome of TAMs isolated from mouse breast tumor was performed. Compared with macrophages from normal spleen, 40 miRs candidates, in 150 TAMs-high and 169 TAMs-low expressed miRs which were selected from miRNome, were further identified by real-time PCR. Importantly, miR-100 was found with TAMs-high expression pattern(about 70 folds changes). Moreover, we found that miR-100 overexpressed RAW264.7 cells and peritoneal macrophages gained M2 phenotype such as CD206+ cells percentage. Mechanism study demonstrated that miR-100 up-regulated IL-1ra expression detected by cytokine array. Furthermore we found that IL-1ra could enhance 4T1 breast cancer cell stemness properties including increasing tumor cell sphere formation and drug resistance in vitro.

Taken together, our results demonstrate that TAMs-high expressed miR-100 could function to maintain the phenotype of TAMs via targeting mTOR pathway, and promote tumorigenesis by enhancing IL-1ra secretion, which would also serve as a promising therapy target to remodel TME and tumor metastasis.

Citation Format: Wei Wang, Yan Liu, Jian Guo, Huiwen He, Junling Xie, Chong Chen, Yunping Luo. MicroRNA-100/mTOR/IL-1ra signaling maintains TAMs phenotype and enhances tumor cell stemness property in mouse breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 724.
Abstract 726: Involvement of proinflammatory chemokines in triple-negative breast cancer (TNBC)
Authors: Ignacio Rosa Mistica C. , Gibbs Carla , Lee Eun-Sook , Son Deok-Soo
Published: July 2016

Objective: Triple-negative breast cancer (TNBC) accounts for 12-24% of total breast cancer, and contributes to the more aggressive, therapy-resistant and poorer outcomes. Our analysis based on The Cancer Genome Atlas (TCGA) indicated that basal-like BC subtype representing TNBC has enriched expression profiles for epidermal growth factor receptor 1 (EGFR) and transforming growth factor alpha (TGFá) compared to other subtypes. In addition, TNBC has a higher proinflammatory index compared to non-TNBC. Here we report that the proinflammatory chemokines such as CXCL1-3 and 8 are highly expressed in human TNBC cells and might be potentiated by TGFá-EGFR axis that contributes to cancer progression of TNBC.

Methods: To gauge the role of TGFá-EGFR axis as a proinflammatory driver in TNBC cells, we first checked the expression profiles for EGFR family such as EGFR, HER2, ErbB3, and ErbB4 between TNBC (MCF10A, MB468, MB231, and BT549) and non-TNBC cells (MCF7, T47D). In addition, the expression levels of downstream signaling for EGFR (Akt/Erk) were measured by western blot. To dissect the role of TGFá-EGFR axis in enhancing proinflammatory index in TNBC cells, we utilized a more comprehensive study of the chemokines and chemokine receptors using human chemokine PCR array. Finally, to further confirm the role of TGFá-EGFR axis in inflammatory burden and cancer progression in TNBC, we used EGFR inhibitor and its downstream targets.

Results: TNBC cells expressed more proinflammatory chemokines such as CXCL1-3 and 8 compared to non-TNBC cells. TNBC cells (MB468, MB231, and BT549) showed higher expression of EGFR which led to higher Akt activation as compared to non-TNBC cells. However, Erk is only activated in MB231. To dissect the involvement of Akt, expression of Akt isoforms in TNBC cells was accessed and Akt1 is the predominant isoform expressed in TNBC cells. To further assess the involvement of Akt1 in proinflammatory burden in TNBC cells, we used a commercial siRNA of Akt1 to knockdown Akt1. Knockdown of Akt1 significantly reduced the CXCL2 promoter activity. In addition, afatinib (an EGFR inhibitor) and MK2206 (an Akt inhibitor) significantly reduced the CXCL2 promoter activity.

Conclusion: Thus, higher expression of proinflammatory chemokines CXCL1-3, and 8, via TGFá-EGFR axis-activated Akt, contributes to the inflammatory burden, probably promoting cancer progression, thereby followed by the overall high mortality in TNBC compared to non-TNBC.

Citation Format: Rosa Mistica C. Ignacio, Carla Gibbs, Eun-Sook Lee, Deok-Soo Son. Involvement of proinflammatory chemokines in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 726.
Abstract 73: Exploring arrayed synthetic CRISPR for functional genomic screening
Authors: Tan Jenille M. , Martin Scott E.
Published: July 2016

Over the past decade, RNAi has been the workhorse for loss-of-function genomic screening. Although powerful, RNAi is hampered by a high rate of false positives driven by off-target effects. New screening technologies based on the CRISPR/Cas9 system are being rapidly adopted, and appear to be significantly less prone to off-target effects. CRISPR/Cas9 screens are conducted in pooled formats for positive and negative selection. However, this format is not amenable to many assay types (e.g., protein translocation assays). In an effort to expand and achieve a more comprehensive approach to functional genomics, we explored the use of arrayed CRISPR/Cas9 screening with synthetic CRISPR RNAs. Testing was performed by reverse transfecting synthetic CRISPR reagents into HCT 116 cells stably expressing Cas9, thereby making the workflow analogous to high-throughput siRNA screening. We first explored and optimized the system with reagents targeting genes associated with DNA replication, including geminin (GMNN). Loss of GMNN is known to result in enlarged nuclei due to aberrant DNA replication, which is easily observed and quantitated by automated microscopy. Analysis of individual cells by microscopy also enables the identification of effects that are only observed in a fraction of the population. After optimizing transfection and assay conditions, we observed CRISPR-induced phenotypes similar to siRNA knockdown. Surprisingly, these effects were observed within 72 hours and affected the vast majority of cells. These effects were even more pronounced in some clonal populations of HCT 116 Cas9. Loss of GMNN was confirmed by immunofluorescence, western blot, and enzyme mismatch cleavage assay. We then expanded this approach to other genes with similar success. By testing and validating the utility of synthetic CRISPR on cells already harboring Cas9, we show that arrayed synthetic CRISPR screening can be a valuable new tool for hit validation and screening.

Citation Format: Jenille M. Tan, Scott E. Martin. Exploring arrayed synthetic CRISPR for functional genomic screening. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 73.