bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1426: Functional analysis of membrane bound versus soluble RANKL: implications for cancer-induced osteolysis.
Authors: McGuire Jeremy J. , Futakuchi Mitsuru , Lynch Conor
Published: August 2014

The primary site of prostate cancer metastasis is the skeleton with the resultant metastases containing areas of extensive osteogenesis and osteolysis. The latter is a process controlled by multinucleated osteoclasts and receptor activator of nuclear kappa B ligand (RANKL) is critical for osteoclastogenesis. RANKL is a type II transmembrane molecule that forms a trimer on the cell surface and mediates its effects via RANK. We have shown that matrix metalloproteinases-3 and -7 (MMP-3, MMP-7) can process RANKL to a soluble form, sRANKL. The objective of this study is to determine the functionality of membrane bound RANKL versus sRANKL in promoting osteoclastogenesis.

Full-length murine RANKL cDNA was isolated and inserted into the pcDNA 3.1(+) expression construct. In vitro translation experiments demonstrated that RANKL was being generated and could be processed by MMP-3 and MMP-7. N-terminal amino acid sequencing revealed that the site of cleavage occurs in the juxtamembrane region of RANKL between amino acids 145 and 146. We hypothesized that cleavage in the juxtamembrane region could generate an active soluble form of sRANKL. Osteoclastogenesis and resorption assays with myeloid precursors demonstrated the functionality of sRANKL. To determine whether RANKL or sRANKL is the major mediator of osteoclastogenesis in vivo we have generated RANKL mutants that are resistant to cleavage by MMPs and other proteases. Using a site directed mutagenesis kit we have designed a number of mutants and found that Δ158, (deletion of amino acids 71-157) and, Δ148, deletion of amino acids 72-147), were resistant to proteolytic cleavage. We anticipate that the while Δ158 and Δ148 are resistant to cleavage, they will be functional. We will therefore test the functionality of these two deletion mutants in-vitro by co-culturing myeloid precursor cells with Cos-7/Cos-1 cells expressing wild type and mutant RANKL proteins.

In conclusion, we have demonstrated that RANKL is proteolytically cleaved to generate a soluble active form. We have generated non-cleavable mutant forms of RANKL that will allow us to assess the role of membrane bound versus soluble RANKL in-vitro and in-vivo. The results of these experiments will further elucidate the roles of RANKL and sRANKL in prostate to bone metastases in addition to being informative for the engineering of future RANKL based therapies.

Citation Format: Jeremy J. McGuire, Mitsuru Futakuchi, Conor Lynch. Functional analysis of membrane bound versus soluble RANKL: implications for cancer-induced osteolysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1426. doi:10.1158/1538-7445.AM2013-1426
Abstract 1427: CCL9 mediates TGF-β regulation of tumor cell survival and lung metastasis.
Authors: Yan Hangyi , Yang Li
Published: August 2014

Cancer metastasis is an ominous feature of tumor progression and accounts for over 90% of cancer-associated deaths. Tumor cell survival in the distant organ is the rate-limiting step in the metastatic process. Primary tumors modulate the pre-metastatic organs and build up a tumor favorable “soil” for the “seeds” (tumor cells). However, the precise cellular and molecular mechanisms remain to be elucidated.

We found that Gr-1+CD11b+ cells or myeloid derived suppressor cells (MDSCs) infiltrate and landscape the pre-metastatic lung into an inflammatory, proliferative, and immune suppressive environment. Tumor cells co-cultured or co-injected with MDSCs showed increased survival and metastasis. Using a cytokine protein array screening, we found that CCL9 (MIP1-γ, macrophage inflammatory protein-1 gamma) is highly secreted in the supernatant of co-culture as well as the metastatic lung. We identified that MDSCs, particularly Ly6C+CD11b+ and F4/80+CD11b+ subsets, are the main source for CCL9 expression. Interestingly, deletion of transforming growth factor -β receptor II (TβRII) in myeloid cells, results in significantly decreased lung metastasis and CCL9 expression. The mechanisms underlying CCL9 regulation of metastasis is currently under investigation.

Citation Format: Hangyi Yan, Li Yang. CCL9 mediates TGF-β regulation of tumor cell survival and lung metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1427. doi:10.1158/1538-7445.AM2013-1427
Abstract 1435: Myeloid TGF-β signaling regulates Platelet factor 4 expression that is important in host immune suppression and tumor metastasis.
Authors: Pang Yanli , Zhu Guiquan , Yang Li
Published: August 2014

Myeloid cells are the most abundant hematopoietic cells in the human body and play a crucial role in tumor angiogenesis, invasion, and metastasis. Here we found that deletion of the gene encoding TGFβ receptor 2 (Tgfbr2) specifically in myeloid cells reduced the level of platelet factor-4 (PF4) in the myeloid cells of 4T1 tumor bearing mice. This is accompanied by decreased lung metastasis of 4T1 mammary carcinoma. Interestingly, deletion of Cxcr3, encoding the receptor for PF4, significantly decreased the lung metastasis of 4T1 mammary carcinoma. The decreased metastasis may attribute to augmented anti-immunity. First, myeloid cells with Cxcr3 deletion showed decreased expression of IL-10, IL-4, iNOs, and arginase. In addition, there was an increased number of both CD4+ and CD8+ T cell in Cxcr3-/- mice compared to the wilt type controls. Our results suggest that TGFβ regulation of PF4 may modulate cancer metastasis through suppressing host anti-tumor immunity.

Citation Format: Yanli Pang, Guiquan Zhu, Li Yang. Myeloid TGF-β signaling regulates Platelet factor 4 expression that is important in host immune suppression and tumor metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1435. doi:10.1158/1538-7445.AM2013-1435
Abstract 1436: TNF-alpha-dependent tumor reprogramming and promotion by microenvironment: Potential involvement of endothelial cells.
Authors: Khodarev Nikolai N. , Pitroda Sean P. , Weichselbaum Ralph R.
Published: August 2014

Interactions of tumor clones with the host microenvironment have profound effects on tumor growth, response to radio/chemotherapy and formation of metastases. TNFα is a potent factor driving and orchestrating such interactions. We used mice with disrupted TNFα receptor I/II signaling to investigate phenotypic and genomic changes induced in tumors by TNFα-deficient or wild type (WT) host microenvironment. At the basal level, disruption of host stromal TNFα signaling resulted in the over-expression of tumor genes involved in extracellular matrix remodeling and angiogenesis. Phenotypically, these tumors exhibited a reduced vascular density, which induced the expression of oxidative stress signaling pathways and increased levels of tissue hypoxia and necrosis. These alterations were paralleled by the suppression of genes involved in chromosomal stability and key effectors of the ATM-dependent/ non-homologous end-joining pathways, which resulted in greater DNA double-strand breaks and caspase 3 activation following IR treatment. We further investigated tumor-associated endothelial cells (TAEC) obtained from tumors grown in WT and TNFα RI/II-/- animals. Differentially expressed genes (DEGs) primarily represented TNFα/Type I IFN-signaling. These genes significantly separated cohorts of patients with chronic inflammation (RA, IBD and liver cirrhosis) from normal samples. We trained these genes in human cancer datasets and defined a six-gene inflammatory signature that predicted significantly reduced overall survival in breast cancer, colon cancer, lung cancer, and glioma. Consistent with these findings, conditioned culture media from human endothelial cells stimulated by pro-inflammatory cytokines accelerated the growth of human colon and breast tumors in immunodeficient mice as compared with conditioned media from untreated endothelial cells. Based on these studies we came to two important conclusions. First, the host stroma may determine phenotypic and transcriptomic properties of tumor cells, and TNFα might be an important component of such microenvironment-dependent tumor reprogramming (MDTR). Second, endothelial cells may be an important cellular component of MDTR and tumor promotion.

Citation Format: Nikolai N. Khodarev, Sean P. Pitroda, Ralph R. Weichselbaum. TNF-alpha-dependent tumor reprogramming and promotion by microenvironment: Potential involvement of endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1436. doi:10.1158/1538-7445.AM2013-1436
Abstract 1468: Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) from patients with small cell lung cancer (SCLC) show heterogeneity in epithelial to mesenchymal transition (EMT) phenotype and evidence of vasculogenic mimicry (VM).
Authors: Metcalf Robert , Booth Jessica , Swain Rajeeb , Bagley Steven , Morris Karen , Krebs Matthew , Hughes Andrew , Brady Gerard , Blackhall Fiona , Dive Caroline
Published: August 2014

Background: Circulating tumour cells (CTCs) and circulating tumour microemboli (CTM) are prevalent in patients with aggressive small cell lung cancer (SCLC). Heterogeneity in epithelial and mesenchymal phenotypes in CTCs and within CTM was investigated given evidence in preclinical models of epithelial and mesenchymal cell co-operativity advantageous for hematogenous spread and the role of epithelial to mesenchymal transition (EMT) in invasion and metastasis.

Methods: Four colour immunofluorescence assays were developed and applied to tumour cells enriched by filtration from peripheral blood of extensive stage SCLC patients prior to treatment. CTCs/CTM were assessed for expression of cytokeratins (CKs, epithelial marker) and vimentin (mesenchymal marker) with DAPI stained nuclei and negative selection of leukocytes and circulating endothelial cells (CECs) by CD45 and/or vascular endothelial-cadherin (VE-cadherin, CD144) expression respectively.

Results and Conclusions: CTCs were identified in all patients that expressed epithelial and/or mesenchymal markers. Individual CTM were detected comprised of tumour cells of heterogeneous EMT status and CTM were also observed containing leukocytes and/or CECs. There was substantial heterogeneity in CK and vimentin expression in CTCs and within CTM consistent with EMT plasticity and with co-operativity between epithelial and mesenchymal tumour cells. A subpopulation of CTCs co-expressed CKs and VE-cadherin that intriguingly, raises the possibility that vascular mimicry accompanies and may facilitate hematogenous spread in this aggressive tumor type. Further studies are required to explore this new hypothesis.

Citation Format: Robert Metcalf, Jessica Booth, Rajeeb Swain, Steven Bagley, Karen Morris, Matthew Krebs, Andrew Hughes, Gerard Brady, Fiona Blackhall, Caroline Dive. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) from patients with small cell lung cancer (SCLC) show heterogeneity in epithelial to mesenchymal transition (EMT) phenotype and evidence of vasculogenic mimicry (VM). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1468. doi:10.1158/1538-7445.AM2013-1468
Abstract 1478: The role of ERBIN in suppressing migration in colon cancer.
Authors: Stevens Payton D. , Yang Mike , Gao Tianyan
Published: August 2014

The LAP (leucine-rich repeat (LRR) and PDZ domain) family protein, ERBIN, is basolaterally localized protein that has previously been identified as a regulator of epithelial cell polarity and an ERBB2/HER2 binding protein. In addition, ERBIN inhibits RAF activation by disrupting by the RAS/RAF interaction. However, the functional importance of ERBIN in cancer remains elusive. In this study, we examined the role of ERBIN in regulating colon cancer progression and metastasis. Stable colon cancer cell lines, including SW480, LIM2405, and Caco2, were generated using lentiviral shRNA to deplete endogenous ERBIN expression. Our results showed that knockdown of ERBIN induced epithelial-mesenchymal transition (EMT)-like phenotype as marked by spindle-like morphology, a decrease in E-cadherin and an increase in vimentin expression. Moreover, the overall rate of cell migration was significantly increased in ERBIN knockdown cells as determined using Transwell assays. When tracking the movement of individual cells using live cell imaging, we found that ERBIN knockdown cells were considerably more motile with faster migrating speed when compared to control cells. Mechanistically, knockdown of ERBIN expression resulted in an increase in both Akt and ERK signaling activation. Taken together, we have identified a novel role of ERBIN in suppressing EMT and cell motility in colon cancer cells.

Citation Format: Payton D. Stevens, Mike Yang, Tianyan Gao. The role of ERBIN in suppressing migration in colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1478. doi:10.1158/1538-7445.AM2013-1478
Abstract 1482: Caveolin is related to progression of malignant transformation in patients with breast tumors.
Authors: Bostanci Hasan , Sahin Tevfik Tolga , Dikmen Kursat , Albayrak Aynur , Yildirim Volkan , Kargici Hulagu
Published: August 2014

Aim: Caveolin seems to be an important factor in tumorigenesis and progression. It has a dual role in both tumor suppression and progression. The proponents of tumor suppression suggest that caveolin is the major protein of the cavea in the cells and these regions accumulate the growth stimulatory signals from certain growth factors such as epidermal growth factor family. Locus of caveolin gene on chromosome 7 is closely related to other tumor suppressor genes. Proponents of the oncogenic role state that caveolin is highly expressed in cancer tissues such as prostate, lung, and renal cell carcinoma when compared to normal counterparts. This type of expression in breast cancers has been ill defined in previous studies. We designed a detailed study in order to evaluate the expression of caveolin in breast cancers and investigation of its possible use as target of specific cancer therapy. The initial phase of the study consists of evaluation of caveolin in patients with benign and malignant breast disease with respect to normal controls. Here we present our preliminary results of the first phase of the study.

Materials and methods: Breast tissue from 202 benign breast tumors, 98 breast cancers and 20 normal controls (obtained from reduction mammoplasties with informed consent) in our center. Caveolin was evaluated by immunohistochemistry.

Results: Caveolin distribution was higher in malignant tissues when compared to control tissues and benign tumors. Furthermore caveolin antigen distribution was enhanced with more aggressive tumor behavior. We observed enhanced caveolin expression especially in myoepithelial cells. The benign tumors expressed higher caveolin when compared to normal controls. In both benign and malignant breast tumors the caveolin expression increased with stromal content of the tumor.

Conclusion: Tumor microenvironment influences the tumor tissue interms of proliferation and metastasis. Caveolin seems to be expressed in the stromal component of the tumor and seems to have increased expression with aggressive nature of the tumor. Results of this preliminary study suggests that caveolin plays a determinative role in tumorigenesis in breast cancer and furthermore correlates with the aggressiveness of the tumor. Caveolin may exert its oncogenic effects by promoting epithelial to mesenchymal transition. Therefore targeting caveolin may be logical strategy in breast cancer treatment.

Citation Format: Hasan Bostanci, Tevfik Tolga Sahin, Kursat Dikmen, Aynur Albayrak, Volkan Yildirim, Hulagu Kargici. Caveolin is related to progression of malignant transformation in patients with breast tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1482. doi:10.1158/1538-7445.AM2013-1482
Abstract 1483: Bmi1 is required for Twist1-mediated breast cancer cell invasion.
Authors: Lin Yu-Huey , Jing Guihua , Liu Junjun
Published: August 2014

Metastasis is the spread of cancer cells from the site of primary tumors to distant organs and the establishment of secondary tumors there. It is a multistep process, and the initial step is cancer cell invasion during which cancer cells move into tissues surrounding the tumor and the vasculature. Transcription factor Twist1 has been identified as a key regulator of metastasis in a murine breast tumor model as suppression of Twist1 expression in metastatic mammary carcinoma cells specifically inhibits their ability to metastasize, and overexpression of Twist1 induces cell motility and promotes an epithelial-mesenchymal transition (EMT). Previous studies have shown that Twist1 works together with polycomb group protein Bmi1 to promote EMT. Here we show that in breast cancer cells, Bmi1 acts downstream of Twist1 to promote the cancer cell invasion. Suppression of Bmi1 in breast cancer cells expressing high level Twist1 reduces cell motility, and ectopic expression of Bmi1 in Twist1-knockdown cells promote cell invasion. The results delineate a novel pathway that leads to cancer cell invasion and metastasis, which may benefit the development of novel therapeutic approaches to treat advanced breast cancer.

Citation Format: Yu-Huey Lin, Guihua Jing, Junjun Liu. Bmi1 is required for Twist1-mediated breast cancer cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1483. doi:10.1158/1538-7445.AM2013-1483

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1486: Role of Slug in chemoresistance, invasion and metastasis of CD133-expressing pancreatic cancer.
Authors: Tsukasa Koichirou , Ding Qiang , Yoshimitsu Makoto , Miyazaki Yumi , Obara Toru , Matsubara Shyuichiro , Maeda Koki , Takao Sonshin
Published: August 2014

Background: Pancreatic cancer is a devastating disease despite current development of therapy. Accumulating evidence suggests that cancer stem cells (CSCs) are responsible for recurrence and chemoresistance. CD133, a marker of CSCs in various solid tumors including pancreatic cancer, has been studied for recent decade. However, the role of CD133 is still obscure. Epithelial-mesenchymal transition (EMT), which is the essential process in the wound healing, development and cancer progression, is one of the CSC characteristics. However, the role of EMT in the pancreatic cancer progression is not sufficiently elucidated. Slug is a member of the Snail family of zinc finger transcription factors and plays a role in the EMT. Slug is demonstrated to be overexpressed in numerous cancers, and involved in migration and tumorigenesis. Recent study demonstrated that EMT was directly suppressed by downregulation of the transcription of Slug (Chakrabarti et al, Nat Cell Biol, 2012). In this study, we investigated the relationship between Slug and biological properties in pancreatic cancer cells.

Materials and Methods: We established a highly migratory cell population with endogenous CD133high derived from Capan-1 cells by migration assay system. Over 90% of CD133high cells expressed CD133 by flow cytometer. Consequently, we established CD133knock-down and Slugknock-down by lentiviral transduction method. We used gemcitabine (GEM) for chemosensitivity assay. To examine the role of Slug, we used migration and invasion assay, wound healing assay, DNA microarray, real-time RT-PCR and Western blot.

Results: 1) DNA microarray and real-time RT-PCR revealed higher expression of EMT-related genes, such as Slug and N-cadherin, in CD133high cells compared to CD133knock-down cells, suggesting that CD133knock-down related to downregulation of Slug. 2) Slugknock-down cells showed more sensitive to GEM by MTT assay compared to parental cells (CD133high cells), but no change of CD133 expression. 3) Slugknock-down cells showed less migratory and invasive abilities than CD133high cells. 4) CD133high cells showed more in vivo metastasis compared to CD133knock-down cells using immunodeficient mice. In vivo experiment using Slugknock-down cells is now ongoing.

Conclusion: Our results suggest that Slug plays an important role in chemoresistance, invasion and metastasis.

Citation Format: Koichirou Tsukasa, Qiang Ding, Makoto Yoshimitsu, Yumi Miyazaki, Toru Obara, Shyuichiro Matsubara, Koki Maeda, Sonshin Takao. Role of Slug in chemoresistance, invasion and metastasis of CD133-expressing pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1486. doi:10.1158/1538-7445.AM2013-1486
Abstract 1492: Systems analysis of epithelial-mesenchymal transition and cell invasion identifies functional gene signatures predictive of survival in lung cancer.
Authors: Schliekelman Mark J. , Taguchi Ayumu , Zhu Jun , Dai Xudong , Zhang Qing , Chin Alice , Wong Chee-Hong , Wang Hong , Gazdar Adi , Laird-Offringa Ite , Tewari Muneesh , Thiery Jean Paul , Hanash Samir
Published: August 2014

We have implemented a systems approach to identify molecular features associated with mesenchymal or epithelial phenotypes and cell behavior in 38 lung adenocarcinoma cell lines that encompassed common known genomic alterations that drive the development of lung adenocarcinoma. Cell line characterization based on morphology, invasiveness, motility and cell-cell adhesion was integrated with proteomic, gene expression, microRNA and methylation profiles. Signatures were established from differential expression of transcripts and proteins in subsets of cell lines with mesenchymal and epithelial characteristics as well as high or low invasion and motility. The mesenchymal, invasion and migration signatures had common upregulation of genes or proteins related to cytoskeletal and actin-binding proteins. Sets of 148 and 74 transcripts corresponding to proteins that were overexpressed in mesenchymal cell lines and high invasive cell lines, respectively, were highly predictive of survival in three independent lung adenocarcinoma datasets. Subsets of 39 and 21 transcripts corresponding to cytoskeletal and acting binding proteins in the mesenchymal and invasive signatures were found to also predict survival in the three independent lung adenocarcinoma datasets, pointing to an association between cytoskeletal and actin binding proteins, a mesenchymal phenotype and tumor progression in lung adenocarcinoma.

Citation Format: Mark J. Schliekelman, Ayumu Taguchi, Jun Zhu, Xudong Dai, Qing Zhang, Alice Chin, Chee-Hong Wong, Hong Wang, Adi Gazdar, Ite Laird-Offringa, Muneesh Tewari, Jean Paul Thiery, Samir Hanash. Systems analysis of epithelial-mesenchymal transition and cell invasion identifies functional gene signatures predictive of survival in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1492. doi:10.1158/1538-7445.AM2013-1492
Abstract 1510: Different change of LEF1/TCFs family members in colorectal carcinogenesis.
Authors: Han Wenxiao , Cao Bangrong , Wang Hongying
Published: August 2014

Wnt/β-catenin pathway is activated in many cancers, especially colorectal carcinoma (CRC). LEF1/TCFs family, which includes LEF1, TCF7(TCF1), TCF7L1(TCF3) and TCF7L2(TCF4), are required for β-catenin-induced transcription of target genes which are widely involved in the carcinogenesis. The aims of our study were to investigate the change of the members of LEF1/TCFs family in chronic inflammation associated colorectal carcinogenesis and the possible roles in carcinogenesis. Chronic inflammation-associated CRC was induced with the AOM-DSS mouse model. Gene expression array experiment was performed with the colonic tissue samples which were collected at different time points during the carcinogenesis. Data analysis strongly indicated that Wnt/β-catenin pathway was activated in chronic inflammation-related colorectal carcinogenesis. However, the members of LEF1/TCFs family showed different changes. The mRNA levels of LEF1 and TCF1 increased while the mRNA levels of TCF3 and TCF4 decreased with the formation of CRC, which were confirmed with real-time PCR. Although some of Wnt target genes were significantly up-regulated, such as MMP7 and Axin2, some were down-regulated, such as Nrcam. Most importantly, the members of LEF1/TCFs family showed the same expression profile in the human samples from normal colon, low grade adenoma, high grade adenoma and CRC. But the mRNA levels of the members were not correlated with the grade of CRC. Moreover, almost all of the target genes up-regulated in AOM-DSS model also increased in human CRC. Finally, prognosis analysis showed that up-regulation of LEF1, TCF1 and corresponding target genes were correlated with higher survival rate (p=0.0068). TCF3, TCF4 and the target genes down-regulated in AOM-DSS model were correlated with disease-free survival rate (p=0.03). Taken together, the members of LEF1/TCFs family changed differently in CRC independent of the status of inflammation. Members of LEF1/TCFs family combined with target genes may be potential prognostic biomarkers for CRC.

Citation Format: Wenxiao Han, Bangrong Cao, Hongying Wang. Different change of LEF1/TCFs family members in colorectal carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1510. doi:10.1158/1538-7445.AM2013-1510
Abstract 1512: Role of Epstein-Barr virus in nasopharyngeal carcinoma under metabolic stress.
Authors: Lau VMY , Tsao SW
Published: August 2014

Most nasopharyngeal carcinoma (NPC) biopsies contain a human pathogen named as Epstein-Barr virus (EBV). EBV infection is considered as an early event involved in the pathogenesis of NPC. However, the close association of EBV with NPC is a paradox. EBV infection is common that occurs in ≥95% of the world's population, yet the incidence rate of NPC is rare worldwide and is only seen in Cantonese speaking ethnic groups in Southern Asia. The mechanisms that regulate EBV infection of NPC/pre-malignant NP cells including the events involved in latency and lytic activation (either in vitro or in vivo) are not well-defined. Expressions of latent and lytic EBV genes have been shown to have variable effects on the biology and malignant properties of EBV-infected cells. Hypoxia and nutrient-starvation are common physiological stresses within tumours and may have impact on regulation of latent and lytic EBV infection in NPC cells.

Here we report that the levels of hypoxia-inducible factor, HIF-1α protein, a prime hypoxic-stress responsive marker for mammalian cells, are higher in EBV-infected NPC/pre-malignant NP cell lines under hypoxia than their non-infected counterparts, which may imply an enhanced survival ability of EBV-infected NP cells. The alteration in gene expression profiles of host cells and expression of EBV genes observed in infected cells may be responsible for the stabilization of HIF-1α and vice versa. Moreover, EBV infection enhances the tumour growth rate and size of NPC cells xenografts in nude mice. The main stem of this study is to examine whether there is a differential response between EBV-infected and non-infected NP cells towards various metabolic stresses. Through studying this project, the mechanism and function of HIF-1α stability by EBV infection will be explored, which is expected to help characterizing the role of EBV in transformation of epithelial cells and how EBV infection may enhance NPC cells to survive inside a tumour core.

Citation Format: VMY Lau, SW Tsao. Role of Epstein-Barr virus in nasopharyngeal carcinoma under metabolic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1512. doi:10.1158/1538-7445.AM2013-1512

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1514: Proteomics of human dense breast stroma.
Authors: Fox Jay W. , Mikesh Leann , Aramadhaka Lavakumar R. , Moskulak Christopher A.
Published: August 2014

Dense breast contains glandular and connective tissue. Less dense breasts are mainly comprised of fat tissue. Dense breast tissue itself is a risk factor for breast cancer with the risk being 4-6 fold higher in women with extremely dense breasts compared to fatty breasts. Typically, breast cancer is more likely to develop in the denser areas of the breast. Furthermore, on mammogram, a tumor is more difficult to detect in dense tissue than in fatty tissue because the tumor looks similar to the tissue that surrounds it. Recognition of the important role of the stroma during the progression of breast cancer leads to the possibility of new targets for the prevention or treatment of the primary breast cancer. In addition, clinically useful biomarkers for the early detection of risk for breast cancer in dense breasts could lead to a significant reduction of mortality. In this investigation we describe a detailed analysis of normal and dense breast stroma using laser microdissection in combination with mass spectrometry and immunohistochemistry of normal and dense breast stroma. Using this approach several potential markers for dense breast were identified and compared to stromal markers associated with breast cancer.

Citation Format: Jay W. Fox, Leann Mikesh, Lavakumar R. Aramadhaka, Christopher A. Moskulak. Proteomics of human dense breast stroma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1514. doi:10.1158/1538-7445.AM2013-1514
Abstract 1515: Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer.
Authors: Youn Je-In , Kumar Vinit , Collazo Michelle , Gabrilovich Dmitry I.
Published: August 2014

Myeloid-derived suppressor cells (MDSC) are one of the major factors negatively regulating immune responses in cancer and many other pathologic conditions. These cells consist of two distinct populations: monocytic MDSC (M-MDSC) and polymorphonuclear MDSC (PMN-MDSC). PMN-MDSC is the predominant group of MDSC accumulated in cancer. Under physiologic conditions, the normal counterpart of M-MDSC - Ly6ChiLy6G- inflammatory monocytes (Mon) differentiate in periphery tissues to macrophages (MΦ) and dendritic cells (DCs). In contrast, a large proportion of M-MDSC, in tumor-bearing mice, acquired phenotypic, morphological and functional features of PMN-MDSC. In human, CD14+HLA-DR-/low M-MDSC from patients with multiple myeloma also differentiated into CD66b+ PMN-MDSC. This effect was caused by soluble tumor-derived factors and mediated by transcriptional silencing of the retinoblastoma (Rb) gene. M-MDSC had an amount of Rb1 similar to that in mature myeloid cells (PMN, DCs) while Rb1 was barely detectable in PMN-MDSC. Lack of Rb1 in mice with conditional deletion of the gene promoted an expansion of splenic CD11b+Ly6G+Ly6Clo cells, similar to the phenotype of PMN-MDSC in tumor-bearing mice. Moreover, immunogeneic EG7 tumor grew much faster in Rb1-KO mice than in Rb1-WT littermates. Mon from Rb1-KO mice acquired significantly increased proportion of PMN after in vitro culture compared to Rb1-WT Mon. In contrast, the overexpression of Rb1 substantially reduced the proportion of PMN-MDSC differentiated from M-MDSC. Histone deacetylase 2 (HDAC-2) was directly involved in down-regulation of Rb1 expression. Inhibition of HDAC in MDSC resulted in the increase in their Rb1 expression, abrogated differentiation of M-MDSC to PMN-MDSC, and restored M-MDSC differentiation towards DCs and MΦ. These results suggest that down-regulation of Rb by epigenetic modification plays a major role in accumulation of MDSC in cancer by regulating PMN-MDSC differentiation from M-MDSC. HDAC may be considered as potential targets for therapeutic regulation of MDSC in cancer.

Citation Format: Je-In Youn, Vinit Kumar, Michelle Collazo, Dmitry I. Gabrilovich. Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1515. doi:10.1158/1538-7445.AM2013-1515

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1517: Pancreatic stellate cells and chemoresistant pancreatic cancer cells induce EMT gene expression in normal pancreatic ductal cells.
Authors: Kumar Krishan , Sahai Vaibhav , Ebine Kazumi , Munshi Hidayatullah G.
Published: August 2014

Pancreatic cancer, one of the deadliest of human cancers, is associated with a collagen-rich pronounced stromal reaction. Previously, we showed that the collagen microenvironment increased the motility of human pancreatic ductal epithelial (HPDE) cells (Ottaviano AJ et al, Cancer Research 2006). Since stellate cells are one of the major contributors to the stromal reaction, we examined the effect of stellate cells on HPDE cells. HPDE cells were labeled with cell tracking dye and then co-cultured stellate cells to allow intimate cell-cell contact. HPDE cells were then isolated 72 hours later using FACS and changes in gene expression analyzed by real time PCR. We found that HPDE cells upregulated expression of epithelial-mesenchymal transition (EMT) markers Zeb1, Snail and vimentin. Stellate cells also increased expression of these EMT markers in HPDE cells expression Kras (HPDE-Kras). However, no change in these EMT markers were seen when the HPDE and stellate cells were prevented from direct cell-cell contact using transwell co-culture system. Treatment with TGF-b type I receptor inhibitor SB431542 blocked the effect of stellate cells on HPDE gene expression. Since ductal cells also encounter malignant cells in vivo, we also examined the effect of co-culture of ductal cells with cancer cells. HPDE cells failed to upregulate EMT markers when they were co-cultured with HPAFII/CD18 cell. However, when HPDE cells were co-cultured with chemo-resistant CD18 cells (CD18-CR), HPDE cells upregulated EMT markers. The effect of CD18-CR cells on HPDE gene expression was only observed when there was intimate cell-cell contact. Also, treatment with SB431542 blocked the effect on HPDE gene expression. These results indicate that the tumor microenvironment may affect the behavior of peri-tumoral non-malignant cells and thereby contribute to tumor progression.

Citation Format: Krishan Kumar, Vaibhav Sahai, Kazumi Ebine, Hidayatullah G. Munshi. Pancreatic stellate cells and chemoresistant pancreatic cancer cells induce EMT gene expression in normal pancreatic ductal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1517. doi:10.1158/1538-7445.AM2013-1517
Abstract 1519: Identification and functional analysis of genes involved in cholesterol metabolism that are upregulated in prostate cancer stroma.
Authors: Ashida Shingo , Kawada Chiaki , Inoue Keiji , Shuin Taro
Published: August 2014

Recent studies have highlighted tumor microenvironment as important in carcinogenesis and progression. Especially in prostate cancer (PC), it has been suggested that tumor microenvironment plays an important role in progression, acquisition of androgen independence, and distant metastasis. However, little is known about the genetic basis of human PC microenvironment. To clarify the mechanism of PC progression and metastasis, we investigated cross-interaction of PC epithelial cells and stromal cells by analyzing genome-wide gene expression profiles. We hypothesized that PC cells could affect on stromal cells to change their characteristics into so-called cancer-associated fibroblasts (CAFs), which might contribute to cancer invasion and metastasis. We identified genes that were up-regulated in PC stroma by analyzing gene expression profiles of normal human prostate stromal cells (PrSC) co-cultured with human PC cells (LNCaP), which included some genes involved in cholesterol metabolism, such as 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). HMGCR is the rate-limiting enzyme for cholesterol synthesis and the pharmalogical target for statins. We confirmed the overexpression of HMGCR in PC stroma by immunohistochemistry. Then we knocked down endogenous HMGCR in PC cells by small interfering RNA, which resulted in the significant reduction of PC cell viability. In contrast, HMGCR overexpression stimulated PC cell growth. These results provide clues to the molecular mechanisms underlying PC invasion and metastasis, and suggest candidate genes the products of which might serve as molecular targets for the treatment of PC.

Citation Format: Shingo Ashida, Chiaki Kawada, Keiji Inoue, Taro Shuin. Identification and functional analysis of genes involved in cholesterol metabolism that are upregulated in prostate cancer stroma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1519. doi:10.1158/1538-7445.AM2013-1519
Abstract 1525: Nuclear homeoprotein SIX1 is associated with stage and grade of endometrial carcinoma.
Authors: Wobker Sara E. , Jefferson Wendy N. , Williams Carmen J. , Bae-Jump Victoria L.
Published: August 2014

Introduction: SIX1 is a member of the homeoprotein gene family encoding transcription factors involved in normal embryogenesis. These proteins function in the cell nucleus to regulate transcription of genes that drive differentiation. SIX1 is also aberrantly expressed in numerous human malignancies, including breast, ovarian and cervical carcinomas. In a mouse model, SIX1 initiates transformation of mammary epithelial cells to diverse histologic types of carcinoma. SIX1 has not yet been studied in endometrial carcinoma, which is the most common gynecologic malignancy in the United States. Thus, our goal was to evaluate SIX1 in clinical samples of endometrial carcinoma and determine if there was an association with stage and grade.

Methods: A tissue microarray (TMA) was created using endometrial carcinoma cases (n=55) and benign controls (i.e. proliferative, secretory and atrophic endometrium) (n=12). Endometrioid and serous histologic types were included in the carcinoma cases. The TMA was stained using a rabbit polyclonal anti-SIX1 antibody (Sigma). The TMA was then scored in a blinded manner by a single pathologist, using presence versus absence of nuclear staining for the stromal cells and the epithelial cells. Clinical data was collected on these samples to include height, weight, BMI, grade (FIGO 1, 2 or 3) and stage (1A, 1B-2B, 3A-4B).

Results: Few controls had nuclear SIX1 in epithelial cells while all controls had nuclear SIX1 in stromal cells. In endometrial cancer samples, there was significantly increased nuclear SIX1 staining in the epithelial cells compared to controls, while there was less nuclear SIX1 in stromal cells. This finding was more pronounced with increasing grade and stage of tumor (Table 1).

Conclusions: Nuclear SIX1 expression is associated with increased grade and stage of endometrial cancer. These findings suggest that SIX1 may play a role in the progression of this common cancer and have utility as a marker of more aggressive disease.

Table 1. Percentage of uterine samples with nuclear SIX1 staining in epithelial and stromal cells Grade Stage Epithelial Stromal Epithelial Stromal Control 3/12 (25) 12/12 (100) Control 3/12 (25) 12/12 (100) Grade 1 16/22 (72) 13/22 (59) 1A 5/11 (46) 10/11 (91) Grade 2 15/16 (94) 6/16 (38) 1B-2B 26/31 (84) 13/31 (42) Grade 3 14/17 (82) 7/17 (41) 3A-4B 12/13 (92) 3/13 (23)

Citation Format: Sara E. Wobker, Wendy N. Jefferson, Carmen J. Williams, Victoria L. Bae-Jump. Nuclear homeoprotein SIX1 is associated with stage and grade of endometrial carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1525. doi:10.1158/1538-7445.AM2013-1525
Abstract 1526: Evaluation of the novel tumor suppressor gene, β-Defensin-1, in colorectal cancer cell line models.
Authors: Layton Austin , Bohanes Pierre O. , Zhang Wu , Lenz Heinz-Josef , Labonte Melissa J.
Published: August 2014

Background: Human defensins are antimicrobial linear peptides with broad anti-microbial activity, organized into two classes, α- and β-defensins. Human β-Defensin 1 (hBD-1) is encoded by the DEFB1 gene located on chromosome 8p23 and functions as an antimicrobial peptide involved in the innate immune response. It is most often expressed in epithelial cells, including those in the large intestines. Previous experiments have shown that hBD-1 exhibits tumor suppressor functions in urothelial and kidney cancer cells. Previous evaluation of germline single nucleotide polymorphisms in the promoter region of DEFB1 demonstrated CRC stage specific effects of rs1800972 and rs179946 in predicting time-to-tumor recurrence in CRC patients. This study examined the cancer specific expression of hBD-1 in CRC compared to normal cells and further evaluated the cellular mechanism associated with the effects of hBD-1 expression in CRC proliferation, cell cycle progression and apoptosis.

Methods: Twenty-two patients colon tissue samples were subjected to immunohistochemistry (IHC) specific for hBD-1 to examine its expression in both normal and tumor cells. To gain insight into the effects of hBD-1 expression in CRC, CaCo2, HCT116, HT29 and SW620 CRC cell lines were transfected with pCMV6-XL4-DEFB1, alongside a control empty vector, to compare the effect of hBD-1 overexpression on cell viability and apoptosis at 24 and 48 hr. In addition, recombinant hBD-1 was exogenously added to CRC cells and analyzed for the effect on cell viability.

Results: Examination of hBD-1 expression from patients’ colon tissue samples by IHC demonstrated that hBD-1 expression was present in the normal colon cells, but its expression was lost in CRC cells. Induction of hBD-1 expression in CRC cell lines resulted in a significant change in cell viability, membrane permeability and induction of apoptosis. Further, the exogenous addition of recombinant hBD-1 to CRC cells resulted in a significant reduction in cell number at 48 hr.

Conclusion: These results demonstrate evidence of loss of hBD-1 expression in CRC cells and further provide evidence for hBD-1’s potential role as a tumor suppressor. Further studies are being conducted to determine the pathway of apoptosis that is induced and to better understand the hBD-1 affects in the CRC cells development and progression and further evaluate the potential utility of targeting hBD-1 as a therapeutic strategy.

Citation Format: Austin Layton, Pierre O. Bohanes, Wu Zhang, Heinz-Josef Lenz, Melissa J. Labonte. Evaluation of the novel tumor suppressor gene, β-Defensin-1, in colorectal cancer cell line models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1526. doi:10.1158/1538-7445.AM2013-1526
Abstract 1530: Alveolar macrophage depletion decreases lung cytokines and decreases mouse lung tumor growth.
Authors: Fritz Jason , Tennis Meredith A. , Malkinson Alvin , Dwyer-Nield Lori
Published: August 2014

While anti-inflammatory therapies are being evaluated in lung cancer, the role of specific immune effector cells and their products in lung carcinogenesis is unclear. Alveolar macrophage numbers increase as lung tumors progress, and subsequent changes in macrophage function are linked to increased growth factor production, accelerated tumor proliferation, enhanced angiogenesis, depressed immune response, and metastasis. However, the signaling molecules linking alveolar macrophages to advanced lung tumor growth are not well defined. To examine how macrophages drive lung tumorigenesis, clodronate-encapsulated liposomes were used to specifically deplete alveolar macrophages in mice bearing chemically-induced lung tumors. Treatment efficacy was assessed by evaluating tumor burden, multiplicity, proliferation index and histologic stage. Alveolar macrophage populations decreased to ≤ 50% of control levels during six weeks of liposomal clodronate treatment. This was associated with significantly decreased pulmonary levels of IGF-1, KC, IL-6 and CCL2. The concentrations of these cytokines strongly correlated with decreased macrophage numbers in lung lavage fluid. Tumor burden and Ki-67 index was reduced by 50% in clodronate-treated mice, corresponding with the 50% decrease in macrophage numbers and cytokine content. Therefore, macrophage depletion attenuates lung tumor proliferation in vivo, consistent with our previous observations linking macrophage IGF-1 production and lung tumor proliferation in vitro. These cytokines appear to mediate macrophage-stimulated lung tumorigenesis, and are thus potential therapeutic targets in lung cancer and chronic inflammatory lung disease.

Citation Format: Jason Fritz, Meredith A. Tennis, Alvin Malkinson, Lori Dwyer-Nield. Alveolar macrophage depletion decreases lung cytokines and decreases mouse lung tumor growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1530. doi:10.1158/1538-7445.AM2013-1530