bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5013: A meta-analysis of MSI frequency and race in colorectal cancer
Authors: Ashktorab Hassan , Ahuja Sadhna , Kannan Lakshmi , Llor Xavier , Ellis Nathan , Xicola Rosa M. , Laiyemo Adeyinka O. , Carethers John M. , Brim Hassan , Nouraie Mehdi
Published: July 2016

BACKGROUND: African Americans(AA) are at a higher risk of colorectal cancer (CRC) and some studies report a higher frequency of microsatellite instability (MSI) in their tumors while others report lower frequency compared to Caucasians.

AIM: To determine and evaluate the association of race and clinical factors with MSI rate through meta- analysis.

METHODS: Twenty-two studies out of 15105 (1997-2015) were evaluated after a search in different literature databases, using keywords “colorectal cancer, microsatellite instability”. We used random effect meta-analysis to calculate the MSI frequency in all studies as well as in African American and Caucasian samples. Meta-regression analysis was used to assess the univariate effect of race, gender, age, tumor location and stage on MSI rate.

RESULTS: The overall MSI frequency among CRCs was 17% (95%CI: 15%-19%, I2 = 91%). The MSI rate among racial groups were 12%, 12% and 14% in AAs, Hispanics and Caucasians respectively and was not significantly different. Sub-group analysis of studies with racial information indicates MSI OR (95% CI) of 0.78 (0.58-1.06) for AAs compared to Caucasians.

CONCLUSION: CRCs demonstrate an MSI frequency of 17%. MSI frequency differences between AAs and Caucasians were not pronounced, suggesting other factors contribute to the racial disparity. There is a large variation in MSI rate between different studies. Methodology approaches and biological sources of this variation should be investigated.

Citation Format: Hassan Ashktorab, Sadhna Ahuja, Lakshmi Kannan, Xavier Llor, Nathan Ellis, Rosa M. Xicola, Adeyinka O. Laiyemo, John M. Carethers, Hassan Brim, Mehdi Nouraie. A meta-analysis of MSI frequency and race 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 5013.
Abstract 5026: EGFR copy number alterations in primary tumors, metastatic lymph nodes and recurrent tumors in areca-quid associated oral squamous cell carcinoma
Authors: Huang Shiang-Fu , Chien Huei-Tzu , Cheng Sou-De , Liao Chun-Ta , Wang Hung-Ming , Hsieh Ling-Ling
Published: July 2016

EGFR and downstream signaling pathway plays important roles in the tumorigenesis in oral squamous cell carcinoma (OSCC). Gene copy number alteration is one the mechanism overexpressing the EGFR protein. We previously demonstrated that EGFR gene amplification was related with lymph node metastasis, tumor invasion and perineural invasion. We appraise the hypothesis that the EGFR gene copy number alteration in the primary tumor can predict that in recurrent tumors or in lymph node metastatic foci. Of 167 primary OSCCs, fluorescence in-situ hybridization (FISH) study showed EGFR polysomy in 19 (11.4%) patients and amplification in 33 (19.8%) patients. EGFR gene amplification was related with lymph node metastasis (χ2 trend test: P = 0.018) Of 57 metastatic lymph nodes, 9 (15.8%) had EGFR polysomy and 14 (24.6%) had EGFR gene amplification. The concordance rate of EGFR gene copy number in primary tumor and lymph node metastasis was 68.4% (McNemar test: P = 0.389). Of 41 recurrent tumors, 5 (12.2%) had EGFR polysomy and 5 (12.2%) had gene amplification. The concordance rate of EGFR gene copy number between primary tumor and recurrence tumor was 65.9% (McNemar test: P = 0.510). From our results, we can predict two-thirds of the EGFR gene copy number alteration in the lymph node metastasis or the recurrent tumor from the analysis of primary tumor. In clinical scenario, for OSCC patients that the lymph nodes or the recurrent tumors are unavailable for EGFR gene copy number analysis, studies from the primary tumors could provide part of the EGFR clonal information in the metastatic or recurrent lesions.

Citation Format: Shiang-Fu Huang, Huei-Tzu Chien, Sou-De Cheng, Chun-Ta Liao, Hung-Ming Wang, Ling-Ling Hsieh. EGFR copy number alterations in primary tumors, metastatic lymph nodes and recurrent tumors in areca-quid associated oral squamous cell 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 5026.
Abstract 5036: Elucidating an oncogenic role of an extracellular matrix protein, Agrin, in hepatocellular carcinoma
Authors: Chakraborty Sayan , Hong Wanjin
Published: July 2016

Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. Despite offering poor survival advantage, broad spectrum receptor tyrosine kinase inhibitors are the sole chemotherapeutic treatments for advanced stages of HCC. Hence, there is a pressing clinical need to broaden the scope of HCC treatments through the systematic identification of suitable candidate(s) overexpressed in liver cancer. Therefore, we performed a SILAC based quantitative proteomics of cell surface proteins in HCC lines and identified proteoglycan Agrin to be overexpressed and secreted in HCC. Our systematic cell biological analysis indicated a role of Agrin in maintaining focal adhesion integrity through stimulation of focal adhesion kinase (FAK). We showed that Agrin functions in cellular migration, invasion and tumor growth. However, the exact mechanisms by which Agrin's mediates liver tumorigenesis remain unknown. The present study dissects how Agrin mediated signals from the extracellular matrix are sensed by tumor cells. Our evidences reveal that Agrin binds to its co-receptor(s) Lrp4 and subsequently activates MuSK to mediate oncogenic effects through activating focal adhesions. Additionally, integrins and focal adhesions collaborate closely with Agrin in communicating ECM signals to intracellular components in multiple HCC cell lines, thereby stimulating their invasive, migratory and growth capabilities. More importantly, Lrp4-MuSK as well as integrin-FAK pathways are crucial downstream signaling axes of Agrin's functions in liver tumorigenesis. The signal transduction network initiated by Agrin activates the oncogenic program in liver by negatively regulating tumor suppressor pathways. These novel insights will have profound impact on the possibility of targeting Agrin mediated oncogenesis in HCC.

Citation Format: Sayan Chakraborty, Wanjin Hong. Elucidating an oncogenic role of an extracellular matrix protein, Agrin, 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 5036.
Abstract 5037: Upregulated superoxide dismutase 2 by specificity protein 1 mediates temozolomide resistance in glioma stem cells
Authors: Chuang Jian-Ying , Liu Jr-Jiun , Chou Shao-Wen , Chang Wen-Chang , Chang Kwang-Yu
Published: July 2016

The resisitant mechanism of glioblastoma mutliforme (GBM) to the standard chemotherapy temozolomide (TMZ) can be attributed to methylguanine methyltransferase (MGMT) expression in only half of the clinical cases. For the other half, it is not fully understood. To elucidate the mechanism, MGMT-negative GBM cell lines, U87MG and A172, were used to develop the TMZ-resistant variants. The variants showed reduced reactive oxygen species (ROS) accumulation by TMZ treatment comparing to the parental one. Further analysis of the cells revealed enhanced expression of superoxide dismutases 2 (SOD2), an antioxidative enzyme, as well as the stem cell-like properties. The protein was shown to associate with glioma stem cells (GSC). Notably, inhibition of SOD2 by diethyldithiocarbamate attenuated the stemness properties and the viability of the resistant variants. Further investigation of the promotor binding study suggested specificity protein 1 (Sp1) as the key factor of the SOD2 response. Moreover, Sp1 expression was even enhanced in the spheroid cells and the TMZ-resistant U87MG cell line. Conversely, treatment with Sp1 inhibitor mithramycin A attenuates SOD2 expression and the stemness properties of the resistant variants. In summary, our results suggested a novel role of SOD2 in TMZ resistant mechanism of GBM. Further study of Sp1-SOD2 pathway as a therapeutic target to restore susceptibility of chemotherapy is therefore warranted.

Citation Format: Jian-Ying Chuang, Jr-Jiun Liu, Shao-Wen Chou, Wen-Chang Chang, Kwang-Yu Chang. Upregulated superoxide dismutase 2 by specificity protein 1 mediates temozolomide resistance in glioma 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 5037.
Abstract 5039: Co-targeting polo-like kinase 1(Plk1) and the Wnt/β-catenin signaling pathway in castration-resistant prostate cancer
Authors: Li Jie , Liu Xiaoqi
Published: July 2016

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events and is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the anti-neoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, Axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1 and Plk1 phosphorylation of Axin2 facilitates the degradation of β-catenin by enhancing the binding between GSK3β and β-catenin. Plk1 phosphorylated Axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.

Citation Format: Jie Li, Xiaoqi Liu. Co-targeting polo-like kinase 1(Plk1) and the Wnt/β-catenin signaling pathway in castration-resistant prostate 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 5039.
Abstract 504: Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are superior to CA 15-3 in predicting tumor burden, patients response to treatment and overall survival (OS) rates in metastatic breast cancer patients from Egypt
Authors: Zekri Abdel-Rahman N. , Bahnassy Abeer A.
Published: July 2016

Background: Monitoring of tumor burden in patients with metastatic breast cancer (mBC) is crucial to determine response to treatment. In addition to radiological procedures and serological biomarkers, circulating, tumor cells (CTCs) are now used to assess the tumor burden and patients’ response to treatment. The circulating cell-free tumor DNA (ctDNA) harboring tumor-specific aberrations has not been properly assessed yet.

Methods: 100 Egyptian patients with locally mBC were assessed for CA 15-3 levels, CTCs number by flowcytometry (FCM) confirmed by RT-PCR (CK and mammaglobin expression), and for ctDNA. The results of the three techniques were compared to the radiographic imaging of tumors, patients response to treatment and overall survival (OS) rates. Paraffin blocks for 40 tumor samples, obtained from the studied patients (20 non-responders and 20 responders) were used to detect p53 gene mutations exons 5-9. CA 15-3 levels and CTC numbers were measured at the same time intervals.

Results: Somatic mutations of p53 were detected in 23/40 (57.5%) sequenced tumors. ctDNA showing the identified mutations in tumor samples, was detected in 78 cases (78%) with high dynamic range. CTCs>4/7.5ml blood were present in 57 out of the 78 (73.1%) ctDNA positive cases, and CA 15-3 was detected in 30 (38.5%) cases. Changes in ctDNA levels and CTCs>4 correlated significantly with the tumor burden (p = 0.034), patients response to treatment (p<0.01), and lower OS rates (p = 0.034&p = 0.01; respectively). CTCs number and ctDNA levels showed higher correlation with changes in tumor burden, compared to CA 15-3 (p<0.001 versus p = 0.046). However, ctDNA provided the earliest measure of treatment response in most of the patients (53%).

Conclusions: ctDNA is an informative, highly sensitive and specific biomarker that could be used to monitor tumor burden in mBC. Together with enumeration of CTCs they can predict tumor response and OS in mBC patients with high accuracy.

Citation Format: Abdel-Rahman N. Zekri, Abeer A. Bahnassy. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are superior to CA 15-3 in predicting tumor burden, patients response to treatment and overall survival (OS) rates in metastatic breast cancer patients from Egypt. [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 504.
Abstract 5046: High-throughput chemical screening for sensitization of bladder cancer to gemcitabine and cisplatin chemotherapy
Authors: Kita Yuki , Kobayashi Takashi , Masuda Norihiko , Sawada Atsuro , Matsui Yoshiyuki , Inoue Takahiro , Kamba Tomomi , Ogawa Osamu
Published: July 2016

Background

Gemcitabine and cisplatin chemotherapy (GC) is the current standard regimen for locally advanced and metastatic bladder cancer (BC). Despite a relatively high initial response rate, some cases do not regress (intrinsic resistance) and the remaining cases often show regrowth after initial shrinkage (acquired resistance). To identify novel therapeutic agents for overcoming these resistances, we applied a high-throughput screening of chemicals administered in combination with GC.

Methods and Findings

As a high-throughput screening, 2100 compounds were administered alone or in combination with GC to human BC cell lines (J82, UMUC-3). Cell viability was determined after 3-day incubation and chemicals that enhanced inhibitory effect of GC were screened. The initial screening identified 26 compounds and further validation narrowed them into the most synergistic agent disulfiram (DSF), an FDA-approved drug for alcoholism. Combination index assay showed synergistic effects of DSF with cisplatin but not with gemcitabine in J82, UMUC-3, T24, HT1197 and HT1376 cells. Co-administration of DSF significantly enhanced apoptosis by GC treatment in UMUC-3 and T24 cells, with significant increase of reactive oxygen species (ROS) production. DSF-induced apoptosis was attenuated in the presence of N-acetyl L-cysteine and Trolox, ROS scavengers. Use of DSF in combination with GC (GCD) significantly inhibited tumor growth of UMUC-3 subcutaneous xenograft on athymic mice (by 39% compared with GC alone, p = 0.02). GCD regimen was as tolerable as GC and no significant differences were observed in body weight of treated mice between the two regimen.

Conclusion

Repositioning of DSF to a chemotherapy sensitizer is a promising treatment strategy, which can be translated rapidly in the future.

Citation Format: Yuki Kita, Takashi Kobayashi, Norihiko Masuda, Atsuro Sawada, Yoshiyuki Matsui, Takahiro Inoue, Tomomi Kamba, Osamu Ogawa. High-throughput chemical screening for sensitization of bladder cancer to gemcitabine and cisplatin chemotherapy. [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 5046.
Abstract 5051: Docosahexaenoic acid inhibits cancer cell invasion and migration by modifying focal adhesion complex structure and signaling
Authors: Mouradian Michael , Bains Palvinder K. , Pardini Ronald S.
Published: July 2016

The extracellular matrix (ECM) is responsible for the regulation of many cellular processes, including survival, proliferation, migration and invasion. Cellular-ECM contact induces a coordinated process recruiting functional protein complexes, consisting of scaffold, adapter, linking and docking proteins, to form focal adhesion (FA) structures. Once formed, FA complexes then recruit signaling proteins to activate downstream cellular pathways. Aberrations in FA structure and signaling have shown to contribute to cancer initiation, progression and cellular motility. Studies investigating the molecular mechanisms accounting for FA structure and function in cancer cells have demonstrated unique growth behavior and migratory properties in vitro. While docosahexaenoic acid (DHA), an omega-3 PUFA, has been shown to inhibit growth of a number of cancer cell lines, the effects of DHA on FA dynamics and signaling have not been investigated. In the present study, we show that DHA induced changes in the morphological growth phenotype in cancer cell lines grown in Matrigel™. DHA treatment also inhibited planar cell movement (cell migration) and amoeboid motility (invasion) in breast and lung cancer cell lines. Associated with the observed morphological changes and inhibited growth, were changes in the expression of a number of proteins involved in the FA complex. Upon further characterization, cell line specific changes in protein expression levels and protein localization of FAK, e-cadherin, paxillin and vinculin were observed with DHA treatment, which suggests that DHA is modifying FA complex structure. Additionally, changes in the phosphorylation patterns of major signaling pathways associated with the FA complex, like EGFR, ERK and c-Myc, were also observed with DHA treatment. Overall, these results provide new insight into FA activation and signaling and demonstrate a new mechanism for DHA treatment in cancer.

Citation Format: Michael Mouradian, Palvinder K. Bains, Ronald S. Pardini. Docosahexaenoic acid inhibits cancer cell invasion and migration by modifying focal adhesion complex structure and signaling. [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 5051.
Abstract 5053: Tumor cell clustering - Identification of new regulators
Authors: LOBJOIS Valérie , Ducommun Bernard
Published: July 2016

Cancer cell aggregation is a key process involved at different stages of tumor evolution, during primary tumor growth, metastatic cells dissemination and secondary tumor evolution. Thus, understanding principles that govern and modulate the ability of cancer cells to aggregate might pave the way for new therapeutic opportunities.

We investigated cancer cell aggregation independently of cell migration or matrix adhesion and demonstrated that the cell-cell adhesion protein E-cadherin and the desmosome proteins DSG2 and DSC2 are both involved this process (Saias, 2015). Strikingly, elevated expression of these proteins in primary tumors has also been reported to be associated with blood detection of clusters of highly metastatic circulating tumor cells and poor prognosis (Aceto, 2014). We also reported that Myosin IIa applies a counter force that slows down aggregation indicating that acto-myosin cytoskeleton tension favors aggregation.

To further decipher the regulation of this process, we developed arrays of hydrogel microwells and robust microscopy-based quantification assay to monitor the kinetics of aggregation of tumor cells in the absence of cell - substrate adhesion. We performed a candidate approach and found that several other members of the cadherin superfamily are involved in the aggregation process. We also conducted a small molecule screen to identify other pathways involved in regulating aggregation of tumor cells. We will report unpublished results on the characterization of the molecular bases of aggregation properties in various tumor cell types and on the identification of new regulatory mechanisms that modulate this process.

- Saias L, Gomes A, Cazales M, Ducommun B, Lobjois V. Cell-cell adhesion and cytoskeleton tension oppose each other in regulating tumor cell aggregation. Cancer Res. (2015) 75(12); 1-8

- Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, et al. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 2014;158:1110-22.

Citation Format: Valérie LOBJOIS, Bernard Ducommun. Tumor cell clustering - Identification of new regulators. [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 5053.
Abstract 5055: SPEN stimulates primary cilia formation, cell migration and is associated with metastasis in breast cancer
Authors: Légaré Stéphanie , Chabot Catherine , Basik Mark
Published: July 2016

The primary cilium (PC) is a microtubule-based and non-motile organelle that functions as animal cells’ antennae, regulating a number of signalling pathways, including the mTOR, PDGFR and Wnt pathways, in addition to being essential for activation of the canonical Hedgehog (Hh) pathway. In breast cancer, the PC is a structure that is frequently lost due to transcription- and mutation-driven alterations in cilium-related genes. In a study aiming to delineate the hormone-independent transcriptional program regulated by SPEN, a gene that we have identified as a tumour suppressor gene in estrogen receptor (ER)-positive breast cancers, we found that SPEN was co-expressed (R>0.95) with a number of genes involved in ciliary biology (P = 1.64E-19), including the ciliogenic transcription factors RFX2, RFX3 and FOXJ1. Interestingly, the overexpression of SPEN resulted in the restoration of the PC in T47D cells, which have previously been shown to lack this structure. Consistent with the re-establishment of a PC in T47D cells, SPEN-overexpressing T47D cells had decreased mTOR signalling, hyperactivation of PC-dependent pathways, including the Hh and PDGFR pathways, and increased migration. Silencing SPEN expression in the non-tumorigenic MCF10A cells, which express high endogenous levels of SPEN and show a high prevalence of PC (36%), considerably decreased PC levels in MCF10A cells and was accompanied with increased mTORC1 activity as well as attenuated cell migration. We also found that SPEN silencing in MCF10A cells decreased the expression of the ciliogenic transcription factor RFX3 and that SPEN interacts with DNA upstream of RFX3 gene transcription start site in chromatin immunoprecipitation experiments. Collectively, these observations indicate that SPEN may mediate its effects, at least in part, through the transcriptional regulation of RFX3. Accordingly, RFX3 expression levels were strongly positively correlated (R = 0.55, P<0.0001) with SPEN levels in a cohort of 82 clinical samples from triple negative breast cancer patients. Notably, high SPEN RNA levels were also predictive of early metastasis in two independent cohorts of 77 (HR = 2.25, P = 0.03) and 170 (HR = 2.23, P = 0.004) ER-negative breast cancer patients. Together, our findings highlight a role for SPEN in the regulation of PC formation and support important functions in the inhibition of mTORC1 and activation of Hh and PDGFR pathways as well as cell migration, a process that may contribute to early metastasis in ER-negative breast cancers.

Citation Format: Stéphanie Légaré, Catherine Chabot, Mark Basik. SPEN stimulates primary cilia formation, cell migration and is associated with metastasis in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5055.
Abstract 5068: Carcinoma-associated fibroblast-derived CXCL12 enhances MDA-MB-231 tumor cell migration though mDia2 formin suppression
Authors: Dvorak Kaitlyn , Eisenmann Kathryn M.
Published: July 2016

The tumor microenvironment (TME) is a heterogeneous region comprised of tumor cells, stromal cells, and secreted factors that make the environment favorable for cancer formation and progression. An important step in the shift to a pro-cancerous environment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are a critical cell type to understand. They are present in a majority of solid tumors and can have a direct effect to enhance adjacent tumor cell motility via their innate ability to secret cytokines, chemokine and growth factors into the TME. We sought to understand how CAF-derived chemokines impact breast tumor cell motility through modification of the F-actin cytoskeleton. We collected cultured media (CM) from WS19T fibroblasts, a patient-derived breast tumor CAF cell line. CAF-CM dramatically enhanced wound-closure in MDA-MB-231 monolayers, relative to normal fibroblast cultured media. The chemokine CXCL12 (SDF1 alpha) has been identified as a potential secreted CAF-directed signal driving tumor cell migration. To determine if CXCL12 was the soluble factor in CAF-CM promoting tumor cell migration, MDA-MB-231 cells were preincubated with AMD3100, an inhibitor of the CXCL12 receptor, CXCR4 prior to CAF-CM application and wounding. AMD3100 effectively blocked CAF-CM-induced MDA-MB-231 migration. CXCL12 incubation with MDA-MB-231 cells equally promoted MDA-MB-231 wound closure, supporting the notion that CXCL12 is secreted by CAFs to promote MDA-MB-231 motility. We previously showed a link between CXCL12 directed CXCR4 signaling and a critical regulator of the dynamic F-actin cytoskeleton, mammalian Diaphanous-related formin (mDia2). This formin regulates the assembly and bundling of unbranched actin filaments and plays a role in tumor cell migration and invasion programs. Western blotting CAF-CM-treated MDA-MB-231 cells revealed a near complete loss of mDia2 protein. mDia1 and ROCK expression were unaffected with CAF-CM treatment. These data were consistent with treatment with a functional mDia FH2-domain inhibitor SMIFH2, which likewise suppresses mDia2 protein levels in MDA-MB-231 cells. It remains unclear if CAF-derived CXCL12 first suppresses mDia2 FH2 activity, leading to loss of mDia2 protein, thus promoting tumor cell motility. Current experiments are designed to address this gap in the knowledge, potentially indicating a role for CAF-derived soluble factors in modulating mDia2 protein function and expression in migrating tumor cells.

Citation Format: Kaitlyn Dvorak, Kathryn M. Eisenmann. Carcinoma-associated fibroblast-derived CXCL12 enhances MDA-MB-231 tumor cell migration though mDia2 formin suppression. [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 5068.
Abstract 5072: Paracrine interactions between Schwann cells and cancer cells promotes perineural invasion via L1cam secretion
Authors: Na’ara Shorook , Gil Ziv , Amit Moran
Published: July 2016

Cancerous neural invasion (CNI) is a well-known route of cancer spread in head and neck cancer. Patients with CNI suffer from severe morbidity due to neuropathic pain and most succumb to cancer within months. The exact mechanism that drives cancer cells to disseminate along nerves remained to be defined. The overall objective of our research was to identify candidate proteins and host cells that contribute to CNI. L1 cell adhesion molecule (L1cam) is a 200kDa transmembrane glycoprotein of the immunoglobulin superfamily. In many human cancers, L1cam is constitutively over-expressed, and its expression is generally associated with poor prognosis and metastases formation. Immunohistochemical analysis of the neural niche in human cancer specimens showed overexpression of L1cam in both cancer cells and Schwann cells. To address the role of L1cam in vitro, we used the Dorsal Root Ganglia. Treatment of Cancer Cells with Anti L1cam mAb resulted in a significant reduction in CNI of cancer cells. Next, we used Schwann cells conditioned media (SCCM) to study the role of soluble Schwann cells derived L1cam. Immunopercipitation of L1cam in SCCM resulted in a significant reduction in L1cam concentration and led to reduced cancer cell migration, invasion, and motility. To study the role of SCCM as a chemo attractant to cancer cells we utilized a modified in-vitro nerve invasion assay that consists of 3 layers Fibroblasts-ECM-Schwann. In this model blocking the activity of L1cam in Schwann cells inhibited the cancer cells invasion through these layers. Finally, in-vivo studies in transgenic mice treated with L1cam mAb triweekly, showed a significant reduction in CNI in the tumors compared to the control group. Taken together, our results identify a paracrine interaction between Schwann cells in the neural niche and the cancer cells promotes perineural invasion via L1cam secretion.

Citation Format: Shorook Na’ara, Ziv Gil, Moran Amit. Paracrine interactions between Schwann cells and cancer cells promotes perineural invasion via L1cam secretion. [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 5072.
Abstract 5079: Sunitinib suppresses ovarian cancer metastasis through the negative modulation of TGF-β-mediated epithelial-mesenchymal transition
Authors: Chen Zibo , Chang Lin-lin , Zhou Tian-yi , Wang Dan-dan , Chen Ying , Zhu Hong , Ying Meidan , Yang Bo , He Qiao-jun
Published: July 2016

The mechanism of the negative regulation TGF-β mediated of epithelial-mesenchymal transition by sunitinib to inhibit ovarian cancer metastasis. We evaluated the inhibitory ability possessed by sunitinib against SKOV3 ovarian cancer cells motility by wound-healing and transwell assays. The results showed that sunitinib inhibited the migration of SKOV3 cells. The results obtained from wound-healing and transwell assays showed that sunitinib suppressed the motility of ovarian cancer SKOV3 cells in a concentration-dependent manner. In another,the effects of sunitinib on TGF-β-induced E-cadherin protein levels was assessed by western-blot, qRT-PCR and immunofluorescence assay. As a result, sunitinib attenuated the downregulation of E-cadherin protein level induced by TGF-β. Western-blot, qRT-PCR and immunofluorescence analyses displayed that the TGF-β stimulation reduced E-cadherin protein level, which could be attenuated by sunitinib. Our recent work reveals that the protein level of snail and the transcriptional activity of smad in sunitinib-treated cells were examined by western-blot and SBE-luciferase assay. Our results imply that sunitinib abolished TGF-β-induced upregulation of snail protein and decreased the transcriptional activity of smad complexes. The western-blot analyses revealed that sunitinib inhibited the upregulation of snail protein level caused by TGF-β treatment. The SBE reporter was constructed by linking the Smad-binding elements promoter upstream of luciferase reporter gene. A remarkable increment of transcriptional activity of Smads complexes was observed in SKOV3 cells exposed to TGF-β, which was significantly prohibited by sunitinib. The current study explores the prohibition of EMT, illustrates the anti-metastatic activities of sunitinib and the underlying mechanisms.

[Keywords] Sunitinib; EMT; TGF-β; E-cadherin; Snail; Smad signal pathway

Citation Format: Zibo Chen, Lin-lin Chang, Tian-yi Zhou, Dan-dan Wang, Ying Chen, Hong Zhu, Meidan Ying, Bo Yang, Qiao-jun He. Sunitinib suppresses ovarian cancer metastasis through the negative modulation of TGF-β-mediated epithelial-mesenchymal transition. [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 5079.
Abstract 5084: Real-time, quantitative cellular analysis of migration and invasion
Authors: Roddy Meagan , Rauch John , O’Clair Lindy , Appledorn Daniel M.
Published: July 2016

Chemotactic-driven cell migration is an important biological process impacting many areas of life science research including immunology, cancer and developmental biology. The dominant high-throughput solution for measuring cellular chemotaxis in vitro is the Boyden-chamber. However, traditional Boyden chamber products have inherent liabilities including: a) the inability to directly visualize the cell migration process, b) a requirement of tens of thousands of cells per well, c) the need to label cells for quantitation, d) a relative insensitivity to surface integrin signaling, and e) high random migration artifacts. Microfluidic solutions have sought to eliminate some of these deficiencies, specifically by providing the ability to visualize the cell migration process, but at the expense of relatively low throughput and cumbersome quantitation methods. Using a live-cell imaging approach and real-time image analysis quantitation, we have developed a new technology which eliminates most of the deficiencies of existing Boyden chambers and most importantly, provides direct visualization of both migration and invasion as it occurs. Integrated metrics precisely quantify the chemotactic response using significantly fewer cells (500-5000 cells per well) in a 96-well format. We exemplify this novel image based technique by studying the migration and invasion of both non-tumor and tumor-derived cell lines. Using Fetal bovine serum (FBS) as a chemoattractant, HT-1080 and NIH-3T3 cells were evaluated for their ability to directionally migrate across a coated surface or invade through a basement membrane extract by embedding the cells in an extracellular matrix prior to seeding. Qualitatively, we observed significant morphological differences between migrating and invading cells, the latter adopting a mesenchymal morphology reminiscent of cells that have undergone epithelial-mesenchymal transition (EMT) and exhibiting thinner, spindle-like morphology as they invade through the matrix. Using both cell count and cell area metrics, we observed a significant difference between migration and invasion kinetics of the relatively non-invasive cell line, NIH-3T3, and the highly invasive human fibrosarcoma derived cell line, HT-1080. Moreover, we observed significant and selective inhibition of HT-1080 cell invasion in the presence of GM6001, a broad inhibitor of Matrix Metalloproteinases (MMPs) with an IC50 of 25.7 nM. This new, real-time, quantitative cellular analysis technique illustrates a novel method to simultaneously study migration and invasion of tumor cells.

Citation Format: Meagan Roddy, John Rauch, Lindy O’Clair, Daniel M. Appledorn. Real-time, quantitative cellular analysis of migration and invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5084.
Abstract 5087: EMT in cancer cells promotes the extracellular matrix remodeling by cancer-associated fibroblasts
Authors: Neri Shinya , Ishii Genichiro , Ochiai Atsushi , Menju Toshi , Sonobe Makoto , Date Hiroshi
Published: July 2016

Microenvironmental factors in tumor tissue induce epithelial-mesenchymal transition (EMT) in cancer cells. However, it remains unclear how EMT-induced cancer cells affect mechanisms of invasion by cancer-associated fibroblasts (CAFs). In this study, we investigated how EMT-induced cancer cells develop supportive functions of CAFs in the process of local invasion. Using an in vitro collagen invasion assay model, EMT-induced cancer cells invaded the collagen matrix to a greater extent compared to control cells when they were cultured alone. Interestingly, CAFs co-cultured with EMT-induced cancer cells invaded to a greater extent compared to those with control cancer cells. We revealed that platelet-derived growth factor (PDGF)-B was upregulated in EMT-induced cancer cells, and the invasion ability of CAFs decreased by shRNA knockdown of PDGF-B in EMT-induced cancer cells. Treatment of a PDGF receptor inhibitor cancelled the increased invasion abilities of CAFs and EMT-induced cancer cells. Our results indicated that EMT-induced cancer cells enhanced the CAF invasion ability through the secretion of PDGF-BB as well as increased the invasion ability themselves, suggesting that EMT-induced cancer cells in the tumor microenvironment could not only exhibit their increased invasion but also promote CAF invasion which might lead to local invasion of cancer cells.

Citation Format: Shinya Neri, Genichiro Ishii, Atsushi Ochiai, Toshi Menju, Makoto Sonobe, Hiroshi Date. EMT in cancer cells promotes the extracellular matrix remodeling by cancer-associated fibroblasts. [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 5087.
Abstract 509: HER2 genomic amplification in circulating tumor DNA from patients with cetuximab-resistant colorectal cancer
Authors: TAKEGAWA. NAOKI
Published: July 2016

Background: Patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS benefit from epidermal growth factor receptor (EGFR)-targeted therapy. However, patients who are treated with anti-EGFR antibodies will eventually develop the resistance to those agents. We previously found that HER2 amplification was one of the mechanisms conferring resistance to anti-EGFR antibody therapy and could therefore be a potential therapeutic target (Yonesaka et al, STM 2011). The aim of this study was to detect HER2 amplification in circulating tumor DNA (ctDNA) from patients with CRC and acquired resistance to anti-EGFR antibody therapy.

Methods: We analyzed plasma ctDNA using digital polymerase chain reaction (PCR) from 18 patients with CRC, who had been treated with anti-EGFR antibody-based therapy (cetuximab) and subsequently acquired resistant cetuximab. We aimed to obtain at least 100 droplets for HER2 and EFTUD2 to accurately assess the ratio. Digital PCR data were analyzed using the QuantaSoft analytical software package (Bio-Rad). The copy number concentration of each gene (HER2 and EFTUD2) was estimated from the Poisson distribution. HER2 amplification with digital PCR was defined as a HER2 ratio (HER2/EFTUD2 copy number ratio) of 1.25 accordingly (Gevensleven H et al, CCR 2013). HER2 gene copy number was analyzed using fluorescence in situ hybridization also in tumor samples before and after acquisition of resistance to cetuximab-based therapy.

Results: Our data showed various HER2 copy number in plasma ctDNA from CRC patients (median: 1.09; range: 0.94-5.18). 22% (4/18) of patients in the cohort exhibited HER2 amplification. One of these patients was found to be positive for HER2 amplification in matched tumor specimens collected after cetuximab therapy, at which point the

patient had acquired cetuximab resistance, despite being negative for HER2 amplification prior to therapy (pre: 1.14, post: 2.78).

Conclusion: Analysis of plasma ctDNA by digital PCR could be useful for detecting HER2 amplification in patients with CRC who were resistant to anti-EGFR antibody therapy. We are now conducting another, large-scale study for evaluating HER2 amplification by ctDNA in CRC patients using digital PCR.

Citation Format: NAOKI TAKEGAWA. HER2 genomic amplification in circulating tumor DNA from patients with cetuximab-resistant 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 509.
Abstract 5093: Fibulin-5 supports pancreatic tumor growth through inhibition of integrin-induced ROS
Authors: Topalovski Mary , Wang Miao , Moore Zachary , Boothman David , Brekken Rolf
Published: July 2016

Pancreatic ductal adenocarcinoma (PDA) is a highly recalcitrant disease. One factor contributing to chemoresistance is thought to be the desmoplastic response seen in these tumors. Fibronectin (FN) is a major constituent of the desmoplastic reaction of PDA. It has been shown that integrin activation by fibronectin can induce reactive oxygen species (ROS) production by stromal cells. Increased ROS has been observed in many cancers, including PDA. Therefore, we propose that oxidation therapy is an attractive approach to increase chemoresponse in PDA as this strategy takes advantage of the sensitivity of these tumors to further ROS insults. We discovered that the matricellular protein Fibulin5 (Fbln5) is upregulated in PDA and reduces FN-mediated integrin-induced ROS production by competing with FN for binding to α5β1 integrin and blocking integrin-induced ROS production. Mutation of the RGD-integrin binding domain in Fbln5 to RGE abolishes the binding of Fbln5 to the integrin and increases integrin-induced ROS production. Fbln5RGE/RGE (RGE) mice, display reduced tumor growth in implant and genetic models of PDA (KIC and KPC) as a direct consequence of elevated ROS levels in the tumor microenvironment. Furthermore, ROS induction correlates with reduced angiogenesis in tumors from RGE animals and had an additive therapeutic effect when combined with standard therapy. We have identified multiple oxidative stress-responsive genes that are elevated in stromal fibroblasts isolated from tumors from RGE animals. One of the most promising targets is NAD(P)H dehydrogenase (quinone 1) (Nqo1). In support of this observation we found that the level of Nqo1 is significantly higher in RGE fibroblasts compared with WT fibroblasts when plated on FN but not collagen or plastic. Furthermore we have demonstrated that FN-mediated induction of ROS and Nqo1 is dependent on β1 integrin and 5-lipooxygenase (5-LOX). Our studies reveal a unique integrin-based mechanism to enhance ROS production to a level that is specifically toxic to the tumor by exploiting the tumor-specific expression of Fbln5. Current studies are focused on understanding the mechanism by which Fbln5 is induced in PDA.

Citation Format: Mary Topalovski, Miao Wang, Zachary Moore, David Boothman, Rolf Brekken. Fibulin-5 supports pancreatic tumor growth through inhibition of integrin-induced ROS. [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 5093.
Abstract 5105: Exosomes secreted by proinflammatory macrophages modulate breast cancer
Authors: Bednarczyk Robert Bronislaw , Tuli Neha Y. , Ben Rahoma Ghada , Maniyar Rachana , Mittelman Abraham , Tiwari Raj K.
Published: July 2016

Multiple cell types reside within the tumor microenvironment (TME) including cancer stem cells, stromal cells, and inflammatory cells. Cellular crosstalk is initiated between these cells through a variety of secreted biomolecules such as cytokines, growth factors, and exosomes. The role of inflammation and numerous secretory factors within the TME have been implicated in the development and progression of various cancers including breast. In this study, we wanted to evaluate the effect of macrophages (a major constituent of inflammation) on breast cancer development. Our study focused on macrophage secreted exosomes which are membraneous nanovesicles that contain various miRNA, mRNA, and proteins. We hypothesized that macrophage secreted exosomes will modulate breast cancer phenotype. An in vitro cell culture system was used in this study to examine macrophage exosome treatments on breast cancer cell lines MCF-7 (ER+) and MDA-MB 231 (ER-, PR-, HER2/neu-). Human monocytic THP-1 monocytes were activated with a common phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) and conditioned media (CM) was collected following 48 h. Exosomes were isolated from THP-1 CM via the use of an exosome isolation reagent and standard centrifugation. Breast cancer cells were then treated with THP-1 exosomes and cell morphology, proliferation, MEK/ERK and PI3K/AKT signaling pathways, and cell cycle were evaluated. MCF-7 and MDA-MB 231 exhibited round and spindle-like morphologies as well as a significant decrease in proliferation at 24 h treatments. Furthermore, MDA-MB 231 displayed increased p-ERK, while MCF-7 showed increased p-MEK following Western blot analysis. Both cell lines exhibited decreased p-AKT and p-S6 kinase expression. Cell cycle analysis showed that MCF-7 and MDA-MB 231 arrested at G2/M at 24 h exosome treatments. THP-1 exosome treatments led to a significant increase in MDA-MB 231 senescence as determined by a senescence assay looking at senescence-associated beta-galactosidase. Increased p21 and decreased Cdc2/Cyclin B1 was also observed which indicates a G2/M arrest. We conclude that specific macrophage secreted exosomes may induce breast cancer cell senescence and affect breast cancer progression.

Citation Format: Robert Bronislaw Bednarczyk, Neha Y. Tuli, Ghada Ben Rahoma, Rachana Maniyar, Abraham Mittelman, Raj K. Tiwari. Exosomes secreted by proinflammatory macrophages modulate breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5105.
Abstract 5106: Unexpected roles for stromal MMP3 during breast cancer
Authors: Hartland Emilia , Jang Charley , Romero Moreno Ricardo , McGarel Megan , Badve Sunil , Littlepage Laurie
Published: July 2016

Matrix metalloproteinases (MMPs) are a family of proteases that have been shown to contribute to the degradation of components of the extracellular matrix and other substrates to promote cancer progression and metastasis. Stromelysin / matrix metalloproteinase-3 (MMP3) is one member of the matrix metalloproteinase family that is overexpressed in human breast cancer. During breast cancer, MMP3 is expressed primarily by stromal cells but also by cancer cells. Overexpression of MMP3 induces an epithelial-to-mesenchymal transition (EMT), promotes hyperplastic growth, and expands a mammary gland stem cell population. In addition, MMP3 is required for the mammary gland stem cell population. In this study, we determined if stromal MMP-3 is required for breast cancer progression for primary and metastatic lung tumor growth. To study this, we transplanted mammary epithelial cancer cells (V0PyMT) into the mammary glands of MMP3 knockout and heterozygous syngeneic mice. We quantified the tumor burden of both primary and metastastic tumors over time and discovered that elimination of stromal MMP3 increased the tumor burden. The tumor pathologies, proliferation rate, and apoptosis were similar between cancer and not cancer tissue. In contrast, the vasculature density decreased. These data suggest that stromal MMP3 inhibits, but is not required for, the development of the primary breast tumor. However, in contrast to our primary tumor data, we interestingly discovered that stromal MMP3 was required for visible lung metastases. These experiments begin to distinguish between the contrasting roles of stromal and epithelial MMP3 during breast cancer. Our data reveal that MMP3 contributes to the metastatic tumor formation within the lungs. This study will help to elucidate new mechanisms through which MMP3 functions within particular microenvironmental contexts during breast cancer progression.

Citation Format: Emilia Hartland, Charley Jang, Ricardo Romero Moreno, Megan McGarel, Sunil Badve, Laurie Littlepage. Unexpected roles for stromal MMP3 during breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5106.