bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1643: p53 family and oncogenic signaling in cancer metastasis
Authors: Xiao Zhi-Xiong , Lv Tao , Wan Yang , Bergholz Johann , Zhang Yujun
Published: July 2016

The tumor suppressor protein p53 is mutated in over 50% of human cancers. Multiple lines of evidence have shown that mutant p53 not only loses its ability to inhibit tumorigenesis, but also gains pro-metastatic functions. However, the molecular mechanisms underlying these effects are still not totally understood. In this study, we demonstrate that the p53 hotspot point mutant, p53(R273H), potently induces cell spreading, migration, and invasion. At the molecular level, p53(R273H) regulates the DLX2-NRP2 pathway in promoting cancer cell migration in vitro and metastasis in vivo. Interestingly, p53(R273H) strongly inhibits tumor cell growth in vitro and in xenograft nude mice model. In addition, we show that p53-related p63 inhibits Erk2 signaling in regulating cell migration/invasion.

Citation Format: Zhi-Xiong Xiao, Tao Lv, Yang Wan, Johann Bergholz, Yujun Zhang. p53 family and oncogenic signaling in cancer metastasis. [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 1643.
Abstract 1668: Bouvardinis a radiation modulator with a novel mechanism of action
Authors: SU. Tin tin
Published: July 2016

Protein synthesis is essential for growth, proliferation and survival of cells. Translation factors are overexpressed in many cancers and their experimental inhibition curbs cancer growth in preclinical models. Differential regulation of translation also occurs upon exposure to cancer-relevant stresses such as hypoxia and ionizing radiation (IR). The failure to regulate translation has been shown to interfere with recovery after genotoxic stress. These findings suggest that modulation of translation, alone or in conjunction with genotoxins, may be therapeutic in oncology. Yet, only two drugs that directly inhibit translation are FDA-approved for oncology use today. We described before the identification of protein synthesis inhibitor bouvardin in a screen for small molecule enhancers of IR in Drosophila melanogaster. Bouvardin was independently identified in a screen for selective inhibitors of engineered human breast cancer stem cells. Here we report the effect of bouvardin in preclinical models of head and neck cancer (HNC) and glioma, two cancer types for which IR is a key therapy choice. Our data show for the first time that bouvardin blocked translation elongation on human ribosomes and suggest that it did so by blocking the dissociation of elongation factor 2 from the ribosome. Bouvardin enhanced the induction of clonogenic death by IR in HNC and glioma cells, although by different mechanisms. Bouvardin enhanced the anti-tumor effect of IR in HNC tumor xenografts in mice. These data suggest that inhibition of translation elongation, particularly in combination with IR, may be a promising treatment option for cancer.

Citation Format: Tin tin SU. Bouvardinis a radiation modulator with a novel mechanism of action. [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 1668.
Abstract 1674: Tripartite motif containing 28 (TRIM28) promotes breast cancer metastasis by stabilizing TWIST1 protein
Authors: Wei Chunli , Cheng Jingliang , Chen Hanchun , Zhang Dianzheng , Fu Junjiang
Published: July 2016

Regulation of gene expression by TRIM28 is not only by transcriptional repression but also by post-translational regulation. Here we report that TRIM28 promotes breast cancer cell metastasis through TRIM28-TWIST1-EMT pathway. We find that TRIM28 is highly expressed in metastatic cancer cell lines and advanced breast cancer tissues. The expression of TRIM28 and TWIST1 is positively correlated with most of the invasive breast carcinomas. Notably, we find that overexpression of TRIM28 upregulates TWIST1 protein, whereas TRIM28 depletion downregulates TWIST1 protein, indicating that TWIST1 upregulation likely occurs at post-transcriptional level, not at transcriptional level. Overexpression of TRIM28 in BT549 breast cancer cell line promotes cell migration and invasion functionally; conversely, knockdown of TRIM28 reduces TWIST1 protein level, up-regulates E-cadherin and down-regulates N-cadherin, and consequently inhibits cell migration and invasion. Furthermore, Immunoprecipitation and GST pull down reveal that TRIM28 interacts with TWIST1 directly, thereby preventing from proteasomal degradation. Moreover, TWIST1 degradation rate is reduced after cycloheximide blocking protein synthesis through the depletion of TRIM28 in BT549 cells. Taken together, our findings suggest that TRIM28 functionally stabilizes TWIST1 protein, reveals a novel mechanism, and provides an effective therapeutic strategy to target TRIM28 in breast cancer treatments.

Citation Format: Chunli Wei, Jingliang Cheng, Hanchun Chen, Dianzheng Zhang, Junjiang Fu. Tripartite motif containing 28 (TRIM28) promotes breast cancer metastasis by stabilizing TWIST1 protein. [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 1674.
Abstract 1677: CLCA2 interaction with EVA1 promotes mammary epithelial differentiation
Authors: Ramena Grace N.T. , Yin Yufang , Yu Yang , Walia Vijay , Elble Randolph C.
Published: July 2016

CLCA2 is a p53-, p63-inducible transmembrane protein that is frequently downregulated in breast cancer. It is induced during differentiation of human mammary epithelial cells, and its knockdown causes epithelial-to-mesenchymal transition (EMT). To determine how CLCA2 promotes epithelial differentiation, we searched for interactors using membrane dihybrid screening. We discovered a strong interaction with the cell junctional protein EVA1 (Epithelial V-like Antigen 1) and confirmed it by co-immunoprecipitation. Like CLCA2, EVA1 is a type I transmembrane protein that is regulated by p53 and p63. It is thought to mediate homophilic cell-cell adhesion in diverse epithelial tissues. We found that EVA1 is frequently downregulated in breast tumors and breast cancer cell lines, especially those of mesenchymal phenotype. Moreover, knockdown of EVA1 in immortalized HMEC caused EMT, implying that EVA1 is essential for epithelial differentiation. Both EVA1 and CLCA2 co-localized with E-cadherin at cell-cell junctions. The interacting domains were delimited by deletion analysis, revealing the site of interaction to be the transmembrane segment (TMS). The TMS primary sequence of CLCA2 was found to be conserved throughout mammals. Moreover, competitive inhibition of the interaction by ectopic expression of the TMS caused partial EMT, suggesting that the interaction is important for differentiation. A screen for other junctional interactors revealed that CLCA2 was involved in two different complexes, one with EVA1 and ZO-1, the other with beta catenin. Overexpression of CLCA2 caused downregulation of beta catenin and beta catenin-activated genes. Thus, CLCA2 links a junctional adhesion molecule to cytosolic signaling proteins that modulate proliferation and differentiation. These results may explain how attenuation of CLCA2 causes EMT and why CLCA2 and EVA1 are frequently downregulated in metastatic breast cancer cell lines.

Citation Format: Grace N.T. Ramena, Yufang Yin, Yang Yu, Vijay Walia, Randolph C. Elble. CLCA2 interaction with EVA1 promotes mammary epithelial differentiation. [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 1677.
Abstract 1679: Notch4 mediates mesenchymal-epithelial-like transition in melanoma
Authors: BonyadiRad Ehsan , Hammerlindl Heinz , Ravindran Menon Dinoop , Hafner Christine , Herlyn Meenhard , Schaider Helmut
Published: July 2016

In melanoma Notch signaling is a well-established oncogenic factor and presumably driver of disease progression. Here we report that overexpression of the constitutively active intracellular domain of Notch4 (N4ICD) directs a mesenchymal-epithelial-like transition that results in an epithelial, low migratory, invasive and proliferative phenotype with decreased tumorigenicity in-vivo. Investigating the expression profile of 50 EMT markers in N4ICD overexpressing cells revealed an epithelial-like gene signature with major EMT markers, including Vimentin, MMP2, Snail1, Snail2 and Twist1, significantly decreased and several epithelial markers, including E-cadherin, Desmoplakin and Occludin significantly increased. N4ICD suppresses the expression of Snail2 and Twist1 in a non-canonical fashion by inducing transcription factors Hey-1 and Hey-2 which directly bind to promoter regions of Snail2 and Twist1 as determined by EMSA and luciferase assays. N4ICD overexpression also results in increased beta-catenin protein levels exclusively limited to the cytoplasm. Immunohistochemical stainings of primary melanomas and subcutaneous metastasis showed a correlation between Notch4, E-cadherin and beta-catenin expression. Our results demonstrate that overexpression of Notch4 ICD results in the emergence of an epithelial-like phenotype with increased E-cadherin and cytoplasmic beta catenin potentially steering the reformation of adherens junctions. This indicates that Notch4 supports a mesenchymal-epithelial-like transition trapping cancer cells in a low proliferative, invasive and migratory epithelial state, suggesting Notch4 to be a tumor suppressor in melanoma.

Citation Format: Ehsan BonyadiRad, Heinz Hammerlindl, Dinoop Ravindran Menon, Christine Hafner, Meenhard Herlyn, Helmut Schaider. Notch4 mediates mesenchymal-epithelial-like transition in melanoma. [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 1679.
Abstract 1681: DUOX1 expression in lung cancer disrupts pro-oncogenic activation mechanisms and localization of Src and EGFR
Authors: Little Andrew C. , Danyal Karamatullah , Bauer Robert A. , Heppner David E. , Hristova Milena , Dustin Christopher , Habibovic Aida , van der Vliet. Albert
Published: July 2016

Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is associated with epithelial-to-mesenchymal transition (EMT), a key feature of tumor invasiveness and metastasis, and that RNAi-mediated DUOX1 suppression can promote EMT, but the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. Furthermore, nuclear Src/EGFR localization and phosphorylation of EGFR-Y1101 in lung cancers was recently associated with metastatic cell behavior and poor clinical outcome. Preliminary findings in alveolar lung cancer A549 cells, which possesses some EMT-like features, indicate that DUOX1 overexpression redistributes Src localization to the plasma membrane and decreases its nuclear accumulation. Moreover, DUOX1 overexpression in A549 cells also suppressed EGF-stimulated EGFR internalization and nuclear translocation, in association with reduced EGFR phosphorylation on its Src target, Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292 (which has retained DUOX1 expression) promoted EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Further mechanistic studies will be performed to elucidate the molecular mechanisms by which DUOX1 is able to alter these events. Collectively, our findings indicate that DUOX1 silencing in lung cancer may contribute to EMT and/or tumor invasiveness by altering Src/EGFR localization and activation mechanisms.

Citation Format: Andrew C. Little, Karamatullah Danyal, Robert A. Bauer, David E. Heppner, Milena Hristova, Christopher Dustin, Aida Habibovic, Albert van der Vliet. DUOX1 expression in lung cancer disrupts pro-oncogenic activation mechanisms and localization of Src and EGFR. [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 1681.
Abstract 1683: Dual role of semaphorin5A in maintaining epithelial and mesenchymal phenotype in metastatic pancreatic cancer
Authors: Saxena Sugandha , Purohit Abhilasha , Varney Michelle , Batra Surinder K. , Singh Rakesh K.
Published: July 2016

Pancreatic cancer (PC) is a highly aggressive disease and in general has metastasized to distant organs by the time of its clinical diagnosis. Poor five-year survival rate of 7% with an average survival time of 5-8 months highlight the gravity of the situation and necessitate a better understanding of the molecules and mechanisms that lead to metastatic dissemination in PC. One of the key processes that lead to metastasis is the gain of cellular migration ability. Previous reports from our laboratory have identified semaphorin5A (SEMA5A), an axon guidance cue molecule regulating cellular migration, as a putative cell adhesion molecule which is involved in organ-specific homing during PC metastasis. Also, differential expression of SEMA5A was observed in cells derived from metastatic sites in comparison to those arising from primary tumors. On the basis of the these findings, we hypothesize that SEMA5A regulates epithelial to mesenchymal changes necessary to drive cellular migration and further leads to establishment of these cells at secondary sites. We utilized various cellular models such as the L3.3 and L3.6pl cell lines with different metastatic potential, cell lines derived from metastatic sites (Capan-1 and CD18/HPAF) with stable knock down of SEMA5A, and cell lines derived from primary tumors (Panc1) with overexpression of SEMA5A. We evaluated epithelial and mesenchymal markers, in vitro cellular migration and the metastatic potential of these cells. We observed down regulation of E-cadherin expression and re-localization of beta-catenin from the membrane to the nucleus in SEMA5A knockdown cells. Similarly, overexpression of SEMA5A in Panc1 cells resulted in higher E-cadherin expression. We also observed higher SEMA5A and E-cadherin expression in the highly metastatic L3.6pl cells in comparison with L3.3 cells. Furthermore, knock-down SEMA5A in Capan-1 cells resulted in enhanced in vitro cellular migration and higher metastasis when control or SEMA5A knockdown cells were orthotopically implanted in nude mice. Our observations suggest that SEMA5A plays a bi-functional role in mediating epithelial to mesenchymal transition at primary site while regulating mesenchymal to epithelial transition and establishment at secondary sites.

Citation Format: Sugandha Saxena, Abhilasha Purohit, Michelle Varney, Surinder K. Batra, Rakesh K. Singh. Dual role of semaphorin5A in maintaining epithelial and mesenchymal phenotype in metastatic pancreatic 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 1683.
Abstract 1684: Mesothelin regulates epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells
Authors: He Xiaoqing , Wang Liying , Despeaux Emily , Rojanasakul Yon
Published: July 2016

Lung cancer and malignant mesothelioma are two of the most deadly forms of cancers. The prognosis of lung cancer and mesothelioma is poor due to limited treatment modalities and lack of understanding of the underlying disease mechanisms. Mesothelin (MLSN), a plasma membrane differentiation antigen, is expressed at a high level in several human cancers, including 70% of lung cancer and nearly all mesotheliomas. Recent studies have shown that plasma and/or pleural effusion MSLN may be used as a biomarker for certain cancers and malignant pleural mesothelioma. However, the role of MLSN in lung cancer and mesothelioma is relatively unknown. In this study, we found that MLSN plays an important role in controlling epithelial-to-mesenchymal transition (EMT) and stem properties of human lung epithelial and mesothelial cells, which control their tumorigenicity. Firstly, MLSN was found to be highly upregulated in human non-small cell lung cancer tissues and in lung cancer and mesothelioma cells, as compared to normal tissues and non-tumorigenic lung epithelial and mesothelial cells. Secondly, knockdown of MLSN by RNA interference significantly reduced anchorage-independent cell growth, tumor sphere formation, and cell migration and invasion in vitro, as well as tumor formation in vivo. Thirdly, knockdown of MLSN attenuated cancer stem cell marker (ALDH) activity and several EMT markers in lung cancer and mesothelioma cell lines. These findings indicate the essential role of MLSN in EMT and malignant properties of human lung cancer and mesothelioma cells. Since EMT is an important process in tumor progression and metastasis, our finding on the role of MLSN may provide new insights into the disease mechanisms and treatment strategies for aggressive lung cancer and mesothelioma.

Citation Format: Xiaoqing He, Liying Wang, Emily Despeaux, Yon Rojanasakul. Mesothelin regulates epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells. [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 1684.
Abstract 1687: KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A
Authors: Kim Min-Young , Shin Jung-Young , Kim Jeong-Oh , Son Kyoung-Hwa , Kang Jin Hyoung
Published: July 2016

Background & Purpose

In recent, KIF5B-RET gene rearrangement has been discovered as a driver oncogene in non-small cell lung cancers. This fusion gene was demonstrated to be able to induce tumorigenesis in vivo mouse model. Hereby, we investigated how KIF5B-RET fusion gene affect tumor differentiation and which transcription factors and signaling pathway are activated.

Methods

We analyzed cell proliferative activity in transformed HEK293T cells with KIF5B-RET fusion gene (K4) and empty vector (V5) using CCK8 assay. We used light microscopy and H&E staining to observe morphological changes of transformed cells. We evaluated protein and mRNA expressions of four EMT markers (E-cadherin, N-cadherin, Snail, and Twist) and two transcription factors (FOXA2, p-STAT5A) using transcription factor PCR array, Western blot, RT-PCR, immunofluorescence staining.

Results

K4 cells showed decreased cell proliferative activity compared with V5 cells. V5 cells had spreading pattern alike mesenchymal phenotype when their density was lower. However, their morphology changed to epithelial phenotype as they were overgrown with higher density. On the other hands, K4 cells exhibited the island growing pattern similar to epithelial phenotype in lower density of tumor cells, but changed to mesenchymal phenotype as the density of tumor cells gradually increased. On H&E staining, the cytoplasm of K4 cells was much smaller than that of V5 cells. We observed that E-cadherin mRNA was down-regulated, but N-cadherin and Twist were up-regulated in K4 cells. Based on the results of repeated screening assay, we finally selected two transcription factors, FOXA2 and STAT5A, which were significantly overexpressed in K4 cells. In K4 cells, phosphorylated-STAT5A was increased and FOXA2 was decreased compared to V5 cells. In addition, the expression of E-cadherin was reduced, but p-GSK3β, p-AKT, and p-ERK were markedly increased in K4 cells. The pattern of immunofluorescence staining of V5 and K4 cells were similar to the protein expressions of E-cadherin and p-STAT5A in Western blot analysis.

Conclusion

Taken together, FOXA2 and E-cadherin are down-regulated and p-STAT5A is up-regulated in HEK-293T cells stably expressing the KIF5B-RET. Our data suggest that KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A.

Citation Format: Min-Young Kim, Jung-Young Shin, Jeong-Oh Kim, Kyoung-Hwa Son, Jin Hyoung Kang. KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A. [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 1687.
Abstract 1699: Protumoral role of trans fatty acid in colorectal cancer
Authors: Ohmori Hitoshi , Kuwai Toshio , Kitadai Yasuhiko , Fujii Kiyomu , Fujiwara Rina , Nishiguchi Yukiko , Sasahira Tomonori , Kuniyasu Hiroki
Published: July 2016

Trans fatty acids (TFAs) is a recent focus of health problems. TFA is a definitive risk factor for cardiovascular diseases and the death. TFA is also possible risk factor for Alzheimer's disease, obesity, diabetes, fatty liver, and ovulation infertility. Relation of TFA with carcinogenic risk is controversy; however, TFA is reported to increase the risk of breast cancer and prostate cancer. Elaidic acid (EA), a trans form of oleic acid, enhances cancer cell growth, invasion, and anti-apoptotic survival. By animal models, EA promotes tumor growth and metastasis to the lung, liver, and peritoneum. EA induces stemness in cancer cells through transactivation of EFGR via SRC from GPR40/120 as receptors in EA-integrated cholesterol raft. Activated EGFR relays the signals to activate canonical and non-canonical wnt pathway and to inactivate notch1 pathways. EA also increases miR-494, which inhibits cell differentiation through decrease of target genes. Continuous EA feeding with dosage alteration increased cancer cell stemness. EA diminishes the efficiency of 5-fluorouracil by increase of residual cancer stem cells. These findings suggest that TFA is a relevant cancer promoting factor. Decision of removal of TFA from foods by FDA might provide an impact to cancer clinics.

Citation Format: Hitoshi Ohmori, Toshio Kuwai, Yasuhiko Kitadai, Kiyomu Fujii, Rina Fujiwara, Yukiko Nishiguchi, Tomonori Sasahira, Hiroki Kuniyasu. Protumoral role of trans fatty acid 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 1699.
Abstract 1712: Intestinal stem cell integrity is preserved through modulation of endoplasmic reticulum stress by guanylyl cyclase C
Authors: Kraft Crystal , Lin Jieru , Snook Adam , Kim Gilbert , Waldman Scott
Published: July 2016

Intestinal stem cells (ISCs) are a long-lived, dynamic cell population at the base of intestinal crypts that adjust their molecular phenotypes as necessary to meet the maintenance and regenerative needs of the epithelium. The long-life, plasticity and proliferative potential of ISCs underlie the necessity for tight regulation, as these cells have been implicated as initiators of colorectal tumorigenesis. In that context, guanylyl cyclase C (GCC) and its effector, cGMP, regulate intestinal epithelial proliferation, differentiation, and susceptibility to intestinal insult and tumor formation. However, the specific role of this signaling axis in ISCs remains undefined. Here, utilizing transgenic mouse models and immunodetection techniques, we reveal that GCC mediates ligand-dependent cGMP signal transduction in ISCs. Further, the absence of GCC alters expression of key ISC markers, decreasing the active ISC marker Lgr5 and increasing the reserve ISC marker Bmi1. Endoplasmic reticulum (ER) stress, which is typically absent from ISCs and has been associated with inflammation and tumorigenesis, is elevated throughout the crypt base in the absence of GCC. Conversely, administration of cGMP or the chemical chaperone taurourdeoxycholic acid eliminates ER stress and restores the normal crypt ISC phenotype. Finally, ISCs exhibit an impaired regeneration response following irradiation in the absence of GCC, highlighting the importance of this signaling pathway in ISC survival and epithelial repair. Together, these observations suggest that GCC signaling modulates ER stress as part of the essential machinery defending the integrity of the ISC niche.

Citation Format: Crystal Kraft, Jieru Lin, Adam Snook, Gilbert Kim, Scott Waldman. Intestinal stem cell integrity is preserved through modulation of endoplasmic reticulum stress by guanylyl cyclase C. [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 1712.
Abstract 1714: The role of Hedgehog signaling in the regulation of human colon cancer stem cells
Authors: Regan Joseph L. , Staudte Stephanie , Schumacher Dirk , Keilholz Ulrich , Haybaeck Johannes , Lehrach Hans , Yaspo Marie-Laure , Henderson David , Steffen Andreas , Toedling Joern , Lesche Ralf , Schaefer Reinhold , Regenbrecht Christian R. , Mumberg Dominik , Lange Martin
Published: July 2016

Recent data support a hierarchical model of colon cancer in which tumor growth is driven by a subpopulation of cancer stem cells (CSCs) that may also be the source of relapse following treatment. Elucidation of the cellular heterogeneity within a tumor would therefore facilitate better characterization of patient subtypes and lead to more personalized and effective treatments. Here, as part of the OncoTrack* consortium, we report the use of Matrigel-based 3D in vitro models of patient-derived colon cancer for the characterization of molecular pathways important in the regulation of CSC self-renewal and survival.

Cellular subpopulations were isolated from patient-derived 3D-culture models based on expression of the CSC marker aldehyde dehydrogenase (ALDH) and then functionally tested for tumor initiation and self-renewal capacity by limiting dilution serial xenotransplantation. These studies demonstrate CSCs to be ALDH+. ALDH+ and ALDH- cells from 3D-culture and xenograft models were then subjected to RNA sequencing for whole transcriptome analysis.

These analyses demonstrated ALDH+ CSCs cells to be enriched for stem cell associated genes (ALDH1A1, LGR5, BMI1, CD44, CD166), developmental processes (embryonic, tissue development, EMT) and signaling pathways (Wnt signaling). In particular, the Hedgehog signaling pathway was found to be enriched in both 3D in vitro and xenograft models.

The role of Hedgehog signaling in colon cancer remains controversial. It has been reported as a negative regulator of Wnt signaling and to be inactive in colon cancer cells lines. Here, using small molecule inhibitors and lentivirus mediated gene knockdown, we report on the role of Hedgehog signaling in the regulation of colon CSC self-renewal and identify non-canonical Hedgehog signaling as a positive regulator of Wnt signaling.

*The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n°115234.

Citation Format: Joseph L. Regan, Stephanie Staudte, Dirk Schumacher, Ulrich Keilholz, Johannes Haybaeck, Hans Lehrach, Marie-Laure Yaspo, David Henderson, Andreas Steffen, Joern Toedling, Ralf Lesche, Reinhold Schaefer, Christian R. Regenbrecht, Dominik Mumberg, Martin Lange. The role of Hedgehog signaling in the regulation of human colon cancer stem cells. [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 1714.
Abstract 1715: Whole transcriptome analysis of patient-derived 3D in vitro and xenograft models of colon cancer identifies placental genes required for the survival of cancer stem cells
Authors: Regan Joseph L. , Staudte Stephanie , Schumacher Dirk , Keilholz Ulrich , Haybaeck Johannes , Lehrach Hans , Yaspo Marie-Laure , Henderson David , Steffen Andreas , Toedling Joern , Lesche Ralf , Schaefer Reinhold , Regenbrecht Christian R. , Mumberg Dominik , Lange Martin
Published: July 2016

Growing evidence supports a subpopulation of cancer stem cells (CSCs) as both the drivers of tumor growth and the source of relapse following treatment. The identification of genes and pathways required for the survival of CSCs may therefore lead to better treatment outcomes. Here, as part of the OncoTrack* consortium, we report the use of 3D in vitro and xenograft models of patient-derived colon cancer for the identification of novel regulators of CSCs.

Increased aldehyde dehydrogenase (ALDH) activity has been demonstrated to be a marker of normal intestinal stem cells and colon CSCs. We therefore isolated and functionally tested ALDH+ cells for CSC properties using 3D in vitro models and xenografts of patient derived colon cancer. These studies revealed ALDH+ cells to be highly enriched for CSCs. In addition, there was a positive correlation between CSC frequency in the models and tumor grade at time of surgical resection.

ALDH+ CSCs were subjected to RNA sequencing for whole transcriptome analysis. These analyses demonstrated ALDH+ CSCs to be enriched for stem cell associated genes (ALDH1A1, BMI1, LGR5, CD44, CD166), signaling pathways (Wnt, Hedgehog) and developmental (embryonic, placental, tissue development, EMT) and metabolic processes (retinol metabolism, drug metabolism, hypoxia).

Lentivirus mediated gene knockdown was carried out on a panel of genes that were differentially expressed in ALDH+ cells from 3D in vitro models and xenografts. These studies led to the identification of genes required for the self-renewal and survival of colon CSCs. Interestingly, these genes have been reported as important regulators of early embryonic and placental development but have not previously been reported in the regulation of cancer stem cells.

*The research leading to these results has received funding from the European Union's Seventh Framework Program (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n°115234.

Citation Format: Joseph L. Regan, Stephanie Staudte, Dirk Schumacher, Ulrich Keilholz, Johannes Haybaeck, Hans Lehrach, Marie-Laure Yaspo, David Henderson, Andreas Steffen, Joern Toedling, Ralf Lesche, Reinhold Schaefer, Christian R. Regenbrecht, Dominik Mumberg, Martin Lange. Whole transcriptome analysis of patient-derived 3D in vitro and xenograft models of colon cancer identifies placental genes required for the survival of cancer stem cells. [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 1715.
Abstract 1716: The role of CD44v9+ colorectal cancer stem cells in the resistance to cetuximab treatment
Authors: Hung Chia-Nung , Chang Ching-Yu , Hsiao Jou , Wei Wan-Chen , Chao Wei-Ting
Published: July 2016

Recent studies found that conventional therapy combined with cetuximab treatment (EGFR inhibitor) to treat metastatic colorectal cancer, had no improvement of patient survival rate in some population. One of possibilities might be cancer stem cells (CSCs) population resist to cetuximab treatment and modulate metastasis, however, the detail mechanism has not been addressed in colorectal cancer. Thus, our study is to decipher the mechanism of resistance to cetuximab in CD44v9+ specific colorectal cancer stem cells. First, to reveal clinical significance of CD44v9, the tumor samples of colorectal cancer patients were analyzed by immunohistochemistry to evaluate CD44v9 expression status in patients. To further investigate the biological mechanism, single cell analysis in CD44v9+ population of HT29 cells was performed with Fluidigm Biomark™ HD system. The results showed that 67% patients had highly expressed CD44v9 with lower survival rate. In vitro studies showed that when HT29 cells were in 5uM cetuximab treatment for 48 hours, CD44v9, Oct4, CD133, CD24, Lgr5, CSCs markers, were up-regulated in RNA level. Furthermore, CD44v9+/Oct4+ cancer cell population was increased but CD44v9+/pEGFR+ cancer cells were eliminated according to immunofluorescence results. The results of single cell analysis demonstrated that CD44v9+/Oct4+ cancer cells highly expressed proliferation markers consistently with cetuximab treatment. Trans-well migration results showed that CD44v9+/Oct4+ cancer cells migrated collectively via mediating E-cadherin dynamics. Furthermore, in clinical outcome, CD44v9 and E-cadherin co-expression were associated with recurrence and metastasis and decreased survival rate significantly. In conclusion, cetuximab failed to target CD44v9+/Oct4+ cancer cells, which characterized as cancer stem cells and might modulate collective cell migration. This finding suggested that new therapeutic strategy to against CSCs would be the potential clinical treatment in colon cancer.

Citation Format: Chia-Nung Hung, Ching-Yu Chang, Jou Hsiao, Wan-Chen Wei, Wei-Ting Chao. The role of CD44v9+ colorectal cancer stem cells in the resistance to cetuximab 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 1716.
Abstract 1718: Characteristics of carbonic anhydrase 9-expressing cells in the human intestinal crypt base
Authors: Suzuki Yozo , Takahashi Hidekazu , Tanemura Masahiro , Nishimura Junichi , Haraguchi Naotsugu , Ohtsuka Masahisa , Miyazaki Susumu , Uemura Mamoru , Hata Taishi , Mizushima Tsunekazu , Yamamoto Hirofumi , Doki Yuichiro , Mori Masaki
Published: July 2016

Background/Aims: In many tissues, stem cells are sequestered in hypoxic microenvironments. However, little is known about the relationship between hypoxia and the intestinal crypt base, the presumed location of intestinal stem cells. This study investigated the expression of carbonic anhydrase 9 (CA9), a hypoxia-inducible membrane-tethered protein, in normal intestinal crypt base cells and in adenoma and early colorectal cancer.

Methods: Using surgically resected human colorectal cancer specimens, we investigated the expression pattern and functional associations of CA9 in human adult normal intestinal epithelia and in adenoma and early colorectal cancer using immunofluorescence, immunohistochemical staining, flow cytometry, and quantitative real-time polymerase chain reaction.

Results: Almost all slender cells in the crypt base in the ileum were CA9+. In the right and left colon, 25% and 40% of all cells, respectively, were CA9+. Notably, CA9+ cells had eosinophilic structures in their basal cytoplasm. Adenomas and T1 colorectal cancer showed broad CA9 expression. Flow cytometry analysis indicated that CA9+ cells had increased mRNA levels of AXIN2, a Wnt signaling factor.

Conclusion: These observations indicate that CA9 expression in normal crypt base cells is associated with intestinal epithelial stemness. CA9 expression may be involved in the carcinogenesis of colorectal cancer.

Citation Format: Yozo Suzuki, Hidekazu Takahashi, Masahiro Tanemura, Junichi Nishimura, Naotsugu Haraguchi, Masahisa Ohtsuka, Susumu Miyazaki, Mamoru Uemura, Taishi Hata, Tsunekazu Mizushima, Hirofumi Yamamoto, Yuichiro Doki, Masaki Mori. Characteristics of carbonic anhydrase 9-expressing cells in the human intestinal crypt base. [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 1718.
Abstract 1719: CD133 attenuates ROS accumulation via a steady increase in the expression of the cystine/glutamate transporter xCT: Consequence on chemoresistance in hepatocellular carcinoma
Authors: Seo Haengran
Published: July 2016

CD133+ cells in hepatocellular carcinoma (HCC) exhibit cancer stem cell (CSC)-like properties as well as resistance to chemotherapeutic agents and ionizing radiation; however, their function remains unknown. In this study, we tried to define new target and compound for enhancement of chemotherapy efficiency in HCC through elucidation of mechanism of CD133-induced chemoresistance.

Here, we show that CD133+ HCC cells exhibit strong resistance to reactive oxygen species (ROS) via persistent maintenance of the ROS-induced increase in xCT expression that upregulates glutathione (GSH) levels, and thereby play a central role in resistance to liver cancer therapy.

ROS are generally known to act as mediators of apoptosis induced by various anti-cancer drugs. We found that increased CD133 expression enhanced the capacity for ROS defense by enhancing cellular GSH levels, and ablation of CD133 attenuated not only the capacity for defense against ROS, but also chemoresistance, in HCC. Sulfasalazine, a potent xCT inhibitor, impaired the ROS defense system and increased the therapeutic efficacy of anticancer therapies in CD133+ HCC but not CD133−HCC in vivo and in vitro. Furthermore, we found that CD133 sustained the ROS stress-induced increase in the expression of xCT, a cystine/glutamate transporter that plays an important role in maintaining GSH levels, and thereby in preventing cell death via anticancer therapies. These results strongly indicate functional roles for CD133 in ROS defense and in evading anticancer therapies in HCC, and suggest that sulfasalazine, administered in combination with conventional chemotherapy, might be an effective treatment for CD133-targeted liver cancer therapy.

Citation Format: Haengran Seo. CD133 attenuates ROS accumulation via a steady increase in the expression of the cystine/glutamate transporter xCT: Consequence on chemoresistance in hepatocellular carcinoma. [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 1719.
Abstract 1733: Inhibition of embryonic stem cells gene expression in pancreatic cancer: Effects of metformin and indole-3-carbinol
Authors: Lyn-Cook Beverly D. , Joseph Stancy , Word Beverly , Hammons George
Published: July 2016

Pancreatic cancer is among the top five deadliest cancers in developing countries and is rapidly increasing in minority populations, particularly African Americans. Understanding molecular mechanisms involved in the etiology and progression of this cancer can greatly improve patient prognosis. Cancer stem cells have been identified in pancreatic tumors and have been shown to contribute to its progression and resistance to standard treatments. Pancreatic cancer stem cells are subject to regulation by key embryonic stem cell transcription factors that are known to be aberrantly expressed in pancreatic cancer, such as SOX2, OCT4 and NANOG. Overexpression of OCT4, SOX2 and NANOG together or separately led to tumor transformation and tumor metastasis. These stem cell factors are known to promote self-renewal by interacting with other transcription factors. Using a human embryonic stem cell RT2 Profiler gene array, numerous stem cell genes were expressed in pancreatic cancer cell lines (MIAPaca2, Panc1). Both cell lines expressed high levels of SOX2, NANOG, CD44, GATA2, POU5F1 (OCT4) and other genes. Panc1 expressed high levels of SOX 17, while MIAPaCa2 expressed high levels of SOX 3,15 and 17. Treatment of cells with indole-3-carbinol alone at 100 and 200μM inhibited or decreased the expression of SOX2, NANOG, CD44, SOX15 and STAT3. However, treatment with metformin alone increased expression of SOX 15 and STAT3 in Panc1 cells but not in MIAPaCa2. Combination of metformin and indole-3-carbinol inhibited the expression of SOX2, NANOG, STAT3, and CD44. SOX2, POU5F1(OCT4) and NANOG are thought to be important players in various human cancers. OCT4 has been found to be expressed in 69% of PDAC and expression was shown too correlated to status and clinical state. High levels of OCT4 and NANOG has been found to correlate to worse prognosis and furthermore, contribute to multidrug-resistance and metastasis. This study have shown that the dietary agent, indole-3-carbinol alone and in combination with metformin down-regulate critical stem cell genes involved in maintenance of pluripotency and self-renewal of cancer stem cells.

Citation Format: Beverly D. Lyn-Cook, Stancy Joseph, Beverly Word, George Hammons. Inhibition of embryonic stem cells gene expression in pancreatic cancer: Effects of metformin and indole-3-carbinol. [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 1733.
Abstract 1734: Body mass index, physical activity and risk of colorectal cancer in the Korean Multi-center Cancer Cohort (KMCC)
Authors: Cho Sooyoung , Shin Aesun , Park Sue K. , Shin Hai-Rim , Chang Soung-Hoon , Yoo Keun-Young
Published: July 2016

Objectives: To examine the association between body mass index (BMI), physical activity and colorectal cancer risk among Korean adults.

Methods: Data from the Korean Multi-center Cancer Cohort (KMCC) between 1993 and 2005 were analyzed. The study population comprised 12,379 subjects aged above 20 years old. The subjects were followed until December 31, 2011 (median follow-up of 10.1 years). Measured weight and height values was used to calculate BMI and self-reported total time spent for physical activity were used. The Cox proportional hazard model was used to estimate the hazard ratio (HR) and 95% confidence intervals (CI) of BMI and physical activity for colorectal cancer risk.

Results: Men who did moderate physical activity showed a lower risk for colorectal cancer (HR 0.35, 95% CI: 0.19-0.65 for 30-419 minutes compared to who spend less than 30 minutes a week doing moderate activities). We did not find any association between the total time of vigorous activities and muscle-strengthening activities and colorectal cancer risk in both men and women. Men with BMI of 25 or higher showed an increased risk for colorectal cancer compared to men with BMI of 18.5 to 22.9 (HR 1.64, 95% CI 0.94-2.88 for 25.0-29.9 kg/m2; HR 1.64, 95% CI 0.94-2.88 for greater than 30.0 kg/m2).

Conclusions: Moderate physical activities were associated with lower colorectal cancer risk among Korean men.

Citation Format: Sooyoung Cho, Aesun Shin, Sue K. Park, Hai-Rim Shin, Soung-Hoon Chang, Keun-Young Yoo. Body mass index, physical activity and risk of colorectal cancer in the Korean Multi-center Cancer Cohort (KMCC). [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 1734.
Abstract 1752: Dietary fat intake during adolescence and breast density among young women
Authors: Jung Seungyoun
Published: July 2016

BACKGROUND: Lack of association between fat intake and breast cancer risk in cohort studies might be attributed to disregard of temporal effects during adolescence when breasts develop and are particularly sensitive to stimuli. We prospectively examined associations between adolescent fat intakes and breast density.

METHOD: Among 177 women who participated in the Dietary Intervention Study in Children, dietary intakes at ages 10-18 were assessed on five occasions by 24-hour recalls and averaged. We calculated geometric mean and 95% confidence intervals for MRI-measured breast density at ages 25-29 across quartiles of fat intake using linear mixed-effect regression.

RESULTS: Comparing women in the extreme quartiles of adolescent fat intakes, percent dense breast volume (%DBV) was positively associated with saturated fat (mean = 16.4% vs. 21.5%; Ptrend<0.001). Conversely,%DBV was inversely associated with monounsaturated fat (25.0% vs. 15.8%; Ptrend<0.001) and the ratio of polyunsaturated fat to saturated fat (P/S ratio; 19.1% vs. 14.3%; Ptrend:<0.001). When examining intake by pubertal stages,%DBV was inversely associated with intake of polyunsaturated fat (20.8% vs. 16.4%; Ptrend = 0.04), long-chain omega-3 fat (17.8% vs. 15.8%; Ptrend<0.001), and P/S ratio (22.5% vs. 16.1%; Ptrend<0.001) before menarche, but not after. These associations observed with%DBV were consistently observed with absolute dense breast volume but not with absolute non-dense breast volume.

CONCLUSIONS: In our study, adolescent intakes of higher saturated fat and lower mono- and polyunsaturated fat are associated with higher breast density measured approximately 15 years later.

IMPACT: The fat subtype composition in adolescent diet may be important in early breast cancer prevention.

Citation Format: Seungyoun Jung. Dietary fat intake during adolescence and breast density among young women. [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 1752.