bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C18: Matrix metalloproteinase-1 (MMP-1) facilitates intravasation and vascular dissemination of human carcinoma cells by regulating the microarchitecture and permeability of intratumoral vasculature
Authors: Juncker-Jensen Anna , Casar Berta , Rimann Ivo , Zajac Ewa , Kupriyanova Tatyana A. , Deryugina Elena I. , Quigley James P.
Published: August 2014

Patients with squamous cell carcinomas (SCCs) often present with advanced stage disease and face one of the lowest 5-year post-therapeutic survival rates. Among 25 signature genes, Mmp1 has been found to be third best SCC tumor predictor, suggesting a critical role of MMP-1 in SCC progression. Functional role of MMP-1 in SCCs was analyzed in this study by employing a highly-disseminating variant of human epidermoid HEp-3 carcinoma, HEp3-hi/diss. The dissemination capacity of HEp3-hi/diss cells in chick embryo spontaneous metastasis models positively correlated with their high levels of MMP-1 expression. MMP-1-deficiency induced by RNA interference resulted in 70-95% inhibition of HEp3-hi/diss intravasation, concomitant with collapse of the intravasation-sustaining microarchitecture of intratumoral vasculature. Furthermore, we have demonstrated the mechanism whereby HEp3-hi/diss-produced MMP-1 contributes to tumor cell intravasation by proteolytically inducing vascular endothelial permeability and increasing rates of carcinoma cells crossing over vascular barriers during the intravasation step of the metastatic cascade.

Citation Format: Anna Juncker-Jensen, Berta Casar, Ivo Rimann, Ewa Zajac, Tatyana A. Kupriyanova, Elena I. Deryugina, James P. Quigley. Matrix metalloproteinase-1 (MMP-1) facilitates intravasation and vascular dissemination of human carcinoma cells by regulating the microarchitecture and permeability of intratumoral vasculature. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C18.
Abstract C20: CXCR4 antagonist inhibits perineural invasion of adenoid cystic carcinoma in preclinical murine model
Authors: Jeong Woo-Jin , Choi Ik Joon , Park Min-Woo , An Soo-Youn , Jeon Eun-Hui , Paik Jin Ho , Sung Myung-Whun , Ahn Soon-Hyun
Published: August 2014

Background. Perineural invasion is a characteristic feature of salivary adenoid cystic carcinoma (ACC). Expression of CXCR4 is also a characteristic feature of ACC which distinguishes it from other non-neurotropic head and neck cancers. Therefore, CXCR4 may play a role in the perineural invasion of ACC. In this study, we aimed to demonstrate CXCR4 expression in ACC, to identify its association with perineural invasion, and to investigate the impact of CXCR4 inhibitor in vitro and in a murine perineural invasion model.

Materials & Methods. Expression of CXCR4 was assessed in ACC cell lines and in human tissue. The effect of gene knockdown using siRNA and a specific blocker of CXCR4 (AMD3100) were evaluated in vitro. A preclinical perineural invasion model was developed using BALB/c nude mouse. The effect of AMD3100 was evaluated in vivo.

Results. CXCR4 was highly expressed in aggressive strains of ACC in vitro, in the tumor in the animal model, and in the tumor of human tissue. SDF-1 expression was also demonstrated in the nerve of murine and human tissue. Gene knockdown by siRNA and inhibition by a CXCR4 specific inhibitor AMD3100 effectively abrogated invasion but not proliferation of ACC in vitro. The rate of perineural invasion was significantly decreased with AMD3100 treatment in the animal model (71.4% vs 18.8%, P=.009).

Conclusions. CXCR4 is highly expressed in ACC and is associated with perineural invasion. AMD3100, which can effectively diminish perineural invasion of ACC in vitro and in vivo, may have an adjuvant role in the management of ACC.

Citation Format: Woo-Jin Jeong, Ik Joon Choi, Min-Woo Park, Soo-Youn An, Eun-Hui Jeon, Jin Ho Paik, Myung-Whun Sung, Soon-Hyun Ahn. CXCR4 antagonist inhibits perineural invasion of adenoid cystic carcinoma in preclinical murine model. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C20.
Abstract C28: N-Myc interactor: A gatekeeper of malignant progression
Authors: Samant Rajeev S. , Devine Daniel J. , Rostas Jack W. , Metge Brandon J. , Shevde Lalita A.
Published: August 2014

Metastasis is the most lethal attribute of cancer progression. It is becoming increasingly evident that epithelial-mesenchymal transition (EMT) is one of the critical events in the metastatic process of solid tumors. Thus, understanding the molecular signaling that negatively influences EMT is of high importance. Here, we present interesting findings about the novel involvement of N-Myc Interactor (NMI) in restricting EMT.

We observed that NMI expression is significantly compromised in advanced breast malignancies. Restoring its expression restricts malignant activities of breast cancer cell lines in vivo and in vitro. Specifically, exogenous expression reduces invasive and migratory potential of tumor cells along with remarkable morphologic transition towards an epithelial appearance. In addition, several key EMT markers such as TWIST, MMP2, SNAI2 and ZEB2 were reduced along with a concomitant gain of epithelial markers such as E-cadherin and Keratin 18. Conversely, silencing NMI from an immortalized breast epithelial line rendered the cells more mesenchymal. Studies using a variety of additional epithelial cell lines consistently revealed that silencing NMI expression coincided with increased expression of both ZEB2 and SNAI2, while NMI overexpression resulted in decreased expression. These expression profile changes were accompanied with significant changes in cell morphology in both 2-dimensional and 3D culture systems. Thus expression of NMI appears to be necessary and sufficient for maintaining the epithelial phenotype.

TGF-β signaling is one of the key signaling pathways that influence EMT. We observed that NMI negatively regulates SMAD driven transcription. We noticed that NMI is able to contribute to this regulation by positively regulating the expression of the inhibitory SMAD, SMAD7. Overall, our data strongly suggests that loss of NMI may be one of the pivotal events in deregulation of TGF-β/SMAD signaling in invasive breast cancers. This loss leads to increased expression of EMT-inducing transcription factors that drive tumor growth and EMT-dependent malignant progression.

Citation Format: Rajeev S. Samant, Daniel J. Devine, Jack W. Rostas, Brandon J. Metge, Lalita A. Shevde. N-Myc interactor: A gatekeeper of malignant progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C28.
Abstract C29: Association of interferon-induced protein with tetratricopeptide repeats 1 gene family with human papilloma virus infection in patients with oral squamous cell carcinoma
Authors: Yen Ai-Hsin , Lai Kuo-chu , Chen Ying-Chen , Liu Chung-Ji , Chang Kuo-Wei , Lee Te-Chang
Published: August 2014

Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) gene family is one of most responsive interferon-stimulated gene families. Four members have been characterized in humans, namely, IFIT1, IFIT2, IFIT3, and IFIT5. The encoded proteins mediate a variety of protein—protein interactions by tetratricopeptide repeat helix-turn-helix motifs to modulate on cellular functions, such as translation initiation, double-stranded RNA-induced signaling, cell proliferation and migration. We have previously demonstrated that IFIT2 silencing activates atypical PKC signaling and induces epithelial-mescenchymal transition (EMT), cell migration, invasion, and metastasis of oral squamous cell carcinoma (OSCC) cells in cultured or animal models. Our results also showed that IFIT2 is potentially a prognosis biomarker of patients with OSCC. In the present study, we further explore the roles of IFIT1 family proteins involved in the regulatory mechanism of metastasis in OSCC cells. We first confirmed that the proteins of various IFIT1 family members are significantly induced by interferon in a dose-dependent manner. We further characterized the expression of IFIT1, IFIT3, and IFIT5 proteins in patients with OSCC. Our present results revealed that most OSCC tumor tissues expressed a variety of amounts of IFIT2 proteins by immunohistochemical analysis. Although the expression levels of IFIT1 and IFIT3 were relatively lower than IFIT2, we observed a close association between IFIT1 and IFIT3 protein expression in OSCC patient-derived tumor specimens. Numerous studies have pointed out that human papillomavirus (HPV) infection is associated with a high risk of oral cancer. Among these 4 IFIT1 family members, we preliminarily demonstrated a well correlation between HPV E6 and IFIT5 protein expression in tumor specimens of OSCC patients, implying a critical role of IFIT5 in response of HPV infection. Accordingly, our present studies implicate that all IFIT1 family are activated by interferon but each member apparently participates in different cellular activities. Their roles involving in OSCC metastasis warrant our further investigation.

Citation Format: Ai-Hsin Yen, Kuo-chu Lai, Ying-Chen Chen, Chung-Ji Liu, Kuo-Wei Chang, Te-Chang Lee. Association of interferon-induced protein with tetratricopeptide repeats 1 gene family with human papilloma virus infection in patients with oral squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C29.
Abstract C30: Resistance to fluid shear stress is a conserved biophysical property of malignant cells
Authors: Barnes James Matthew , Nauseef Jones T. , Henry Michael D.
Published: August 2014

During metastasis, cancer cells enter the circulation in order to gain access to distant tissues, but how this fluid microenvironment influences cancer cell biology is poorly understood. A longstanding view is that circulating cancer cells derived from solid tissues may be susceptible to damage from hemodynamic shear forces, contributing to metastatic inefficiency. However, there have been few efforts to directly test this hypothesis. Here we report that compared to non-transformed epithelial cells, transformed cells are remarkably resistant to fluid shear stress (FSS) in a microfluidic protocol, exhibiting a biphasic decrease in viability when subjected to a series of millisecond pulses of high (750-6,300 dyn/cm2) FSS. We show that magnitude of FSS resistance is influenced by several oncogenes including myc, ras, and PI3K; is an adaptive and transient response triggered by reparable plasma membrane damage; and requires extracellular calcium and actin cytoskeletal dynamics, including the activity of Rho kinase. This novel property of malignant cancer cells may facilitate hematogenous metastasis and indicates, contrary to expectations, that cancer cells are quite resistant to destruction by hemodynamic shear forces.

Citation Format: James Matthew Barnes, Jones T. Nauseef, Michael D. Henry. Resistance to fluid shear stress is a conserved biophysical property of malignant cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C30.
Abstract C31: Downregulation of IL-12 through IL-6 production increased cancer metastasis after radiotherapy
Authors: Seong Jinsil , Lee Eun Jung , Jeong Keun Young
Published: August 2014

Radiotherapy (RT) is a potent antitumor modality. However, unwanted effect is also reported such as increased metastasis through complex molecular mechanism including cytokines. Cytokines are soluble messengers that directly stimulate immune cells and stromal cells at the tumor site and enhance cancer cells recognition by immune cells. Numerous studies have investigated that cytokines have anti-tumor efficacy for cancer therapy. In this study, we investigated levels of 6 cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α) in serum of before and after RT of HCC patients treated with RT and evaluate its significance in relation to clinical outcome. As a result, intrahepatic metastasis out of RT field increased in patients with IL-12 decreas after RT (p = 0.043).

IL-12 is an inflammatory cytokine that induces interferon-γ production from Th1 and NK cells. IL-12 mainly produced from antigen presenting cells as dendritic cells (DCs) which against many tumors is probably the result of its stimulatory effects on cell mediated immune responses.

Therefore, we investigated anti-tumor and anti-metastatic effects of mesenchymal stem cells expressing IL-12 (MSC/IL-12) treatment with ionizing radiation (IR) in murine hepatic cancer (HCa-1) model. Tumor bearing syngenic mice were treated with IR, MSC/IL-12 or combination of both. Combination of MSC/IL-12 and IR resulted in suppression of lung metastasis as well as significantly growth delay of Hca-1. Especially, expressions of CD4, CD8 and CD11C were increased in tumors of MSC/IL-12 and IR treated group compared with only IR.

Also, to understand the detailed mechanism on anti-tumor effect of IL-12 in IR treated HCC, splenocyte were irradiated with 2, 5 Gy and measured IL-6 and IL-12 by ELISA after cultured. As results, IL-6 significantly increased by IR, while IL-12 increased at 1 day but it was decreased in dose dependent manner at 3days. These results indicated that IL-12 production could be mediated by IL-6 signaling. To prove that, DCs were irradiated with 5 and 10 Gy in presence or absence of LPS after differentiated from mice bone marrow and analyzed expressions of CD80 and 86 by FACS. The expression of CD80 and 86 decreased in irradiated-DCs comparing to no-irradiated DCs. Also, LPS-induced expressions of CD 80, 86 and IL-12 increased in α-IL-6 treated DCs compared with no-treated DCs.

Taken together, diminishing IL-12 via IL-6 production by IR seems to increase the treatment failure after radiotherapy in hepatocellular carcinoma.

Citation Format: Jinsil Seong, Eun Jung Lee, Keun Young Jeong. Downregulation of IL-12 through IL-6 production increased cancer metastasis after radiotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C31.
Abstract C44: Activation of focal adhesion kinase by direct interaction with β4 integrin in an EGFR-Src-dependent pathway in tumor progression
Authors: Tai Yu-Ling , Shen Tang-Long
Published: August 2014

Although β4 integrin and FAK (focal adhesion kinase) are individually known to be associated with tumor progression, only recently have they been suggested to function in a signaling axis. Here, we demonstrate in vivo and in vitro physical and functional interactions between β4 integrin and FAK. An 11-amino acid motif of FAK was identified as essential for interaction with the cytodomain of β4 integrin in malignant cells but not in less malignant cells, implicating the role of this interaction in malignancies. Furthermore, this interaction occurs in an adhesion-dependent manner and results in autophosphorylation of FAK's Tyr-397. EGF signaling and Src activity were indispensable for this interaction. Finally, disruption of the β4 integrin/FAK complexes reduced cell proliferation, migration and invasion as well as tumorigenesis, in concurrence with the decreases in phospho-Akt and phospho-p38. Together, our data elucidate an underlying mechanism for the regulation of tumor progression by the novel β4 integrin/FAK complexes through a physical interaction that is EGF/Src dependent. The significance of this finding is highlighted by often co-overexpression of both integrin β4 and FAK in patients' cancerous tissues with colon cancer revealed by immunohistochemistry analyses. Based on the structural prediction for the complex of integrin β4 and FAK, it may lead us to gain more insight on the anticancer therapies of aggressive cancers.

Citation Format: Yu-Ling Tai, Tang-Long Shen. Activation of focal adhesion kinase by direct interaction with β4 integrin in an EGFR-Src-dependent pathway in tumor progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C44.
Abstract C49: Tissue tranglutaminase regulates β-catenin signaling in ovarian cancer cells
Authors: Condello Salvatore , Cao Liyun , Matei Daniela
Published: August 2014

Background: Tissue transglutaminase (TG2) is a multifunctional enzyme involved in protein crosslinking and cell adhesion to fibronectin (FN). In cancer, TG2 induces epithelial-to-mesenchymal transition (EMT) contributing to metastasis. As cadherins bind β-catenin at cell-cell junctions, disruption of adherens junctions destabilizes cadherin-catenin complexes. The goal of the present study was to analyze whether and how TG2 interacts with and regulates β–catenin signaling in ovarian cancer (OC) cells.

Methods: Western blotting, immuno-fluorescence and immunohistochemistry measured TG2 and β-catenin expression in OC cells and human tumors. B-catenin function was analyzed by TCF/LEF1 promoter reporter assay and Q-RT-PCR based quantification of mRNA levels for target genes (c-myc and cyclin D1). Protein-protein interaction was analyzed by co-immunoprecipitation and confocal microscopy. Cell proliferation was measured by MTT assay.

Results: We observed a significant correlation between TG2 and β-catenin expression levels in OC cells and tumors. TG2 augmented Wnt/β-catenin signaling, as evidenced by enhanced β-catenin transcriptional activity, inducing transcription of target genes cyclin D1 and c-Myc. By promoting integrin-mediated cell adhesion to FN, TG2 associates with and recruits c-Src, which in turn phosphorylates β-catenin at Tyr654, releasing it from E-cadherin, and rendering it available for transcription regulation. Ultimately the TG2—FN interaction stimulates OC cell proliferation by enhancing β-catenin signaling.

Conclusions: Our data show that TG2 regulates β-catenin expression and function in OC cells and define a c-Src dependent mechanism through which this occurs. We posit that the TG2-FN interaction initiates this process and could be a new cancer target.

Citation Format: Salvatore Condello, Liyun Cao, Daniela Matei. Tissue tranglutaminase regulates β-catenin signaling in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C49.
Abstract C5: Study of the relationship between RAR-beta2 hypermethylation and invasive esophageal squamous cell carcinoma
Authors: Wu Ruei-Siang , Yu Chun-Chieh , Li Yue-Yuah , Wu Ming-Tsang , Li Ruei-Nian
Published: August 2014

Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer death among men in Taiwan, and the five-year survival rate is below 10%. The retinoic acid receptor beta2 (RAR-beta2) is frequently deleted or silenced at early stages in tumor progression. Most of RAR-beta2 repressions was associated with promoter hypermethylation in several epithelial carcinomas. The aim of this study is to investigate RAR-beta2 promoter methylation status in different stages of ESCC patients. Through methylation-specific PCR analysis, methylation rate of RAR-beta2 was 21.1% (15/71) in tumor cases, but unmethylated in all normal counterparts. In RAR-beta2 promoter hypermethylation cases, the methylation frequency of lymph nodes invasion and tumor metastasis was twofold increased. Moreover, RAR-beta2 promoter methylation frequency was increased with 14.7% and showed significant decrease survival rate in advanced stage. Esophageal cancer cell lines 81T, 81T 1-1 and 81T 1-4 were treated with 5-aza-dC (DNA methylation inhibitor), the level of RAR-beta2 methylation was decreased in all ESCC cell lines. Moreover, migration ability was inhibited in 81T cell line. The migration ability of 81T 1-1 and 81T 1-4 cell lines were decreased about 50%. Our findings indicate that RAR-beta2 hypermethylation in ESCC could be useful for prognosis marker. RAR-beta2 methylation may become one of the clinical therapy targets and invasion-migration markers.

Citation Format: Ruei-Siang Wu, Chun-Chieh Yu, Yue-Yuah Li, Ming-Tsang Wu, Ruei-Nian Li. Study of the relationship between RAR-beta2 hypermethylation and invasive esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C5.
Abstract C51: c-Abl and Arg are activated in human primary melanomas, promote melanoma cell invasion, proliferation, survival, and drive metastatic progression
Authors: Ganguly Sourik S. , Fiore Leann S. , Sims Jonathan T. , Friend J Woodrow , Srinivasan DivyaMani , Thacker Matthew , Cibull Michael L. , Chi Wang , Plattner Rina
Published: August 2014

Despite 40 years of clinical trials, there has been little improvement in one-year survival rates for patients with metastatic melanoma, and the disease is essentially untreatable if not cured surgically. The paucity of chemotherapeutic agents that are effective for treating metastatic melanoma indicates a dire need to develop new therapies. Here, we found a previously unrecognized role for Abl kinases in melanoma progression. We demonstrate that Abl kinases (c-Abl, Arg) are activated in human melanoma cell lines, in primary melanomas (60%), and in a subset of benign nevi (33%). Using siRNA and pharmacological approaches, we show that c-Abl/Arg activation is functionally relevant because it is required for melanoma cell proliferation, survival, matrigel-invasion and single-cell 3D invasion. Moreover, active c-Abl transcriptionally upregulates MMP-1, and using rescue approaches we show that c-Abl promotes invasion via a STAT3-MMP-1 pathway. Conversely, active Arg drives invasion in a STAT3-independent manner, and upregulates MMP-3 and MT1-MMP, in addition to MMP-1. Paradoxically, c-Abl and Arg also increase expression of TIMP-1, a negative regulator of MMPs, both at the transcript and protein level via a STAT3-dependent pathway. In addition to negatively regulating MMPs, TIMPs also have anti-apoptotic functions, and high TIMP-1 expression is correlated with decreased survival and a poor clinical outcome in breast and colon cancer patients. Currently, we are in the process of testing whether c-Abl and Arg promote survival via induction of TIMP-1. Most importantly, we found that Abl kinases not only promote in vitro processes important for melanoma progression, but also promote metastasis in vivo, as inhibition of c-Abl/Arg with nilotinib dramatically inhibits lung colonization/metastasis in a mouse model using two different melanoma cell lines. Taken together, these data identify Abl kinases as critical, novel, drug targets in metastatic melanoma, and indicate that nilotinib may useful in preventing metastasis in patients with melanomas harboring active Abl kinases.

Citation Format: Sourik S. Ganguly, Leann S. Fiore, Jonathan T. Sims, J Woodrow Friend, DivyaMani Srinivasan, Matthew Thacker, Michael L. Cibull, Wang Chi, Rina Plattner. c-Abl and Arg are activated in human primary melanomas, promote melanoma cell invasion, proliferation, survival, and drive metastatic progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C51.
Abstract C54: Characterization of HGF-mediated EMT and its inhibition with novel HGF pathway inhibitors using transcriptomic analysis
Authors: Tuttle Kevin J. , Burton Jason , Johnson W Evan , Hansen Marc DH
Published: August 2014

The ability of cancer cells to detach from their neighbors, invade through local tissues, and establish distant metastases is a major contributor to cancer mortality. Epithelial-mesenchymal transition (EMT) describes a developmental process by which cells break cell-cell adhesions and gain motility. Interestingly, cells generated by EMT also bear stem cell properties and are more resistant to chemotherapeutics. EMT is triggered by a number of cellular signaling pathways, including via hepatocyte growth factor. Inappropriate signaling in epithelial tumors is thought to drive EMT, facilitating cancer metastasis. A common feature is the activation of a number of transcriptional programs. Here we report the results of transcriptomic studies that are designed to characterize transcriptional changes in cells undergoing EMT in response to HGF stimulation. MDCK cells, a normal epithelial cell line, were stimulated with HGF for different amounts of time under otherwise identical culture conditions. Since cell substrate composition affects the robustness of the HGF response, we performed this experiment on cells grown on different extracellular matrices in an attempt to better understand how cellular substrates provide context to HGF signaling. Triplicate samples of mRNA were generated from these cultures for transcriptomic sequencing and analysis. Each sample generated 20-40 million sequence reads that were assembled into known and de novo (novel) transcripts using Tuxedo Suite, allowing detection of differential transcript expression by cummeRbund and edgeR. Many of the differentially expressed genes correlated well to known cancer pathways. HGF signaling is widely accepted to play an important role in cancer progression and to be an important target for development of novel therapeutics. We previously identified a series of novel HGF pathway inhibitors using a phenotypic screening approach. Compounds were subjected to analysis of their structure-activity relationship, in vivo toxicity, and pharmacokinetic profiles. Compounds that block EMT without affecting proliferation were applied to MDCK cells prior to HGF stimulation and mRNA samples generated from these cultures. We report how these inhibitors alter the transcriptional response of MDCK cells to HGF stimulation, providing a molecular basis for how these inhibitors prevent EMT.

Citation Format: Kevin J. Tuttle, Jason Burton, W Evan Johnson, Marc DH Hansen. Characterization of HGF-mediated EMT and its inhibition with novel HGF pathway inhibitors using transcriptomic analysis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C54.
Abstract C55: Detachment of epithelial cells is driven by myosin-based contractility and RhoA
Authors: Hoj Jacob Peter , Fullmer Kendra , Davis John , Morrell David , Hansen MDH
Published: August 2014

Metastasis requires shedding and migration of individual cells from the tumor. In tumors of epithelial origin, cells often maintain strong cell-cell interactions that must be disassembled prior to cell detachment. Remodeling of cell-cell adhesions occurs during epithelial-mesenchymal transition (EMT), a developmental process where individual cells are triggered to detach and migrate to distant sites by developmental signaling programs. It is thought that inappropriate activation of EMT drives metastasis, particularly by allowing detachment and migration of cancer cells. While much is known about migration and invasion, how cell-cell junctions are detached remains poorly understood. Here we sought to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/ scatter factor-induced EMT. We find that HGF signaling actually increases cell-cell adhesion between cells in suspension, suggesting that changes in cell-cell adhesion are not enough to drive epithelial scattering. Instead, cell-substrate adhesion seems to play a bigger role. Cell-substrate adhesion increases upon HGF stimulation and alterations in the pliability of the subtratum alter cells' ability to undergo scattering. Interestingly, scattering is impaired in cells expressing both active and inactive RhoA mutants. While this also indicates a role for important changes in cell-substrate adhesion during cell-cell detachment, RhoA also affects myosin-based contractility. We sought to examine how myosin-based contractility contributes to cell-cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely initiation of cell spreading and an inability to rupture cell-cell adhesions in response to HGF treatment. Interestingly, when cell spreading is induced by transient treatment of cells with blebbistatin, blebbistatin washout results in cellular events that closely resemble HGF-induced epithelial scattering. This suggests that global suppression and subsequent induction of myosin II activity may be sufficient to rupture cell-cell adhesions. Such experiments also suggest a role for RhoA, as blebbistatin-induced scattering is not fully completed in cells expressing a dominant-negative RhoA mutant. Taken together, these results suggest that cell-cell detachment is controlled by contractility, with a contribution by RhoA-mediated changes in cell adhesion.

Citation Format: Jacob Peter Hoj, Kendra Fullmer, John Davis, David Morrell, MDH Hansen. Detachment of epithelial cells is driven by myosin-based contractility and RhoA. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C55.
Abstract C56: microRNA-200 control of tumor cell cytoskeletal changes and invasion
Authors: Ungewiss Christin , Roybal Jonathon D. , Peng David H. , Gibbons Don L.
Published: August 2014

Lung cancer is the leading cause of all cancer-related deaths, accounting for 160,000 deaths per year in the US, primarily caused by metastasis. Understanding the molecular mechanisms leading to metastasis will identify new therapeutic targets for improved treatment strategies. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we have shown that loss of microRNA-200 family (miR-200) expression is necessary and sufficient to drive an epithelial-to-mesenchymal transition (EMT), allowing cells to acquire an invasive and metastatic phenotype necessary for metastasis. Cell motility is characterized by the reorganization of the actin cytoskeleton, for which we are currently investigating the role of miR-200. Cortactin is a scaffold protein known to play a central role in different signaling pathways involved in the formation of pro-invasive cell structures by recruiting actin-associated proteins and proteases leading to the degradation of the extracellular matrix (ECM). Maturation of cell protrusions requires the phosphorylation of cortactin by upstream tyrosine kinases such as src and arg. Functional assay of cell behavior on 2D agarose demonstrates actin reorganization within our cell lines with different miR-200 levels. Biochemical analysis of cell lines from our model and human lung cancer cells shows that metastatic cells (low-miR-200) have increased levels of phosphorylated cortactin compared to the non-metastatic cells (high miR-200), leading us to hypothesize that miR-200 regulates the biochemical activation of signaling pathways that mediate cortactin phosphorylation and a pro-invasive cytoskeletal reorganization. Inhibition of src using pharmacologic inhibitors and an siRNA approach significantly reduces phosphorylated cortactin levels, inhibits in vitro cell migration and invasion, and decreases the invasiveness of cells grown in 3D, demonstrating the importance of this pathway in invasion. Furthermore, our study demonstrates that the pro-survival PI3K pathway can be inhibited using cortactin siRNA, underlining the multi-faceted role of cortactin in tumor progression.

Citation Format: Christin Ungewiss, Jonathon D. Roybal, David H. Peng, Don L. Gibbons. microRNA-200 control of tumor cell cytoskeletal changes and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C56.
Abstract C57: The role of the microRNA-200 family in the regulation of tumor cell signaling and metastasis in lung cancer
Authors: Roybal Jonathon D. , Peng David H. , Ungewiss Christin , Mandal Pijus K. , McMurray John S. , Byers Lauren A. , Gibbons Don L.
Published: August 2014

Lung cancer is one of the deadliest cancers in the United States due primarily to the development of metastatic disease, resulting in a 16% five year overall survival. Unfortunately response rates are poor with the current modalities used to treat patients, which implies that a greater understanding of the biology of invasion and metastasis is essential. It is thought that the epithelial-mesenchymal transition (EMT) plays a critical role in regulating invasion, migration, and metastasis. The microRNA-200 family (miR-200) is a key maintenance factor for the epithelial phenotype by regulating EMT-inducing transcription factors such as Zeb1, and subsequent metastatic potential, although the mechanistic details of this regulation are incompletely understood. Recent research has demonstrated that miRNAs are capable of regulating the target mRNAs that coordinate intracellular signaling pathways. Using our mutant Kras and p53 murine model of metastatic lung adenocarcinoma, we have shown through reverse phase protein array (RPPA) profiling that in poorly metastatic lung tumor cells, high miR-200 levels can suppress cancer-promoting signaling molecules, such as Signal Transducer and Activator of Transcription 3 (STAT3). In contrast, highly metastatic lung tumor cells with low miR-200 expression have STAT3 activation and increased migratory and invasive properties. Utilizing siRNA approaches or pharmacological inhibition of STAT3 in highly metastatic lung tumor cells decreased their ability to migrate and invade. Our data suggests that the STAT3 pathway is under the control of miR-200/Zeb1 axis and could provide a potential therapeutic target in lung cancer patients.

Citation Format: Jonathon D. Roybal, David H. Peng, Christin Ungewiss, Pijus K. Mandal, John S. McMurray, Lauren A. Byers, Don L. Gibbons. The role of the microRNA-200 family in the regulation of tumor cell signaling and metastasis in lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C57.
Abstract C59: Lipid expression dynamics in epithelial cells undergoing HGF-induced EMT
Authors: Alexander Kristen , Coutu Brendan , Prince John , Hansen Marc
Published: August 2014

Cancer metastasis occurs when cells detach from the primary tumor, invade through local tissues, migrate to distant sites, and colonize new tumors. The morphological change within the cell is termed epithelial-mesenchymal transition (EMT), as cells change from their epithelial state and derive more primitive, mesenchymal characteristics. A cellular signaling pathway that stimulates EMT is initiated by hepatocyte growth factor (HGF), which binds and activates the c-Met receptor tyrosine kinase. Activation of HGF signaling induces dramatic changes in cell morphology and behavior. HGF signaling exerts its effect by altering gene transcription and proteome profiles in cells triggered to undergo EMT. The proteomic changes in HGF-induced cell lines is well documented, however changes in lipid expression and their contribution to EMT are poorly understood. Here we develop and employ mass spectrometry-based approaches to analyze lipid expression changes in cells. This method allows for analysis of thousands of lipids in a single sample and the determination of the relative abundance in each lipid. We have found that seventy lipids undergo dramatic regulatory expression changes in MDCK cells undergoing HGF-induced EMT. This effort could provide important clues into the identity of lipid modification or synthesis programs that are required for EMT and, more broadly speaking, for cellular events associated with cancer progression. Understanding their contribution and function could allow for identification of therapeutic targets for intervention in cellular processes required for cancer metastasis.

Citation Format: Kristen Alexander, Brendan Coutu, John Prince, Marc Hansen. Lipid expression dynamics in epithelial cells undergoing HGF-induced EMT. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C59.
Abstract C64: Leptin increases the metastatic potential of breast cancer cells
Authors: Misrha Ameet , Blackburn Anneke C. , Parish Christopher R. , Wong Ma-li , Licinio Julio
Published: August 2014

In women, obesity is linked to 20% of all cancer deaths. The molecular mechanisms are largely unknown but the risk of invasive/metastatic disease with obesity may include elevated levels of hormones like leptin, IGFs and estrogens. Leptin is a pleiotropic hormone primarily synthesized by adipose tissues and circulating levels are higher in obese people (200ng/ml) than nonobese people (10ng/ml-30ng/ml). We examined the effect and mechanism of leptin treatment on the metastatic and cancer stem cell (CSC) characteristics of human breast cancer cells in vitro. Leptin receptor (LEPR) was found to be highly expressed in more invasive breast cancer cells (29-fold higher mRNA expression in MDA-MB-231 than MCF7, P<0.0001). To assess the effect of leptin treatment on metastatic capacity of breast cancer cells, MCF7 cells were treated for 14 days with leptin. Compared to untreated MCF7 cells, treatment with 200ng/ml of leptin increased breast cancer cell motility (scratch assay, 1.85-fold, P=0.0018) and invasiveness (2.45-fold, P=0.0021). Leptin treatment induced epithelial to mesenchymal transition (EMT), decreasing CDH1 mRNA (0.1-fold, P=0.002) and increasing SNAI2 mRNA (4.1-fold, P<0.0001) compared to the untreated cells. Leptin also induced aldehyde dehydrogenase activity by 6.4 fold (ALDEFLUOR, P=0.0069) and increased colony forming capacity (mammosphere assay, 2.03-fold, P<0.0001) indicating an increased stem-like phenotype. Co-treatment of leptin and neutralizing antibody against TGFB1 abrogated these effects, indicating that the induction of EMT and CSC characteristics was mediated via TGFB1. This study indicates the significance of leptin in contributing to the poor outcomes of breast cancer in obese patients.

Note: This abstract was not presented at the conference.

Citation Format: Ameet Misrha, Anneke C. Blackburn, Christopher R. Parish, Ma-li Wong, Julio Licinio. Leptin increases the metastatic potential of breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C64.
Abstract C76: Axl signaling is required for stem cell traits and metastasis in breast cancer
Authors: Tiron Crina , Pelissier Fanny , Wnuk-Lipinska Katarzyna , Stefansson Ingunn , Virtakoivu Reeta , Miyano Masaru , Sandal Tone , Micklem David , Garbe James , Stampfer Martha , Ivaska Johanna , Akslen Lars , LaBarge Mark , Lorens James
Published: August 2014

Epithelial-to-mesenchymal transition (EMT) endows carcinoma cells with migratory, survival and stem cell-like attributes that facilitate therapeutic resistance and metastasis. Expression of the Axl receptor tyrosine kinase in breast cancer correlates with EMT, poor survival and is prevalent in patient metastases. Axl upregulation in mammary epithelial cells by EMT-transcription factors, TGFbeta or hypoxia, establishes an autocrine-signaling loop with its ligand, Gas6. Inhibition of Axl signaling blocks EMT/ cancer stem cell traits including invasiveness, drug resistance, mammosphere formation, in vivo tumor initiation, and prevents spontaneous metastasis in orthotopic breast cancer models. Congruently, we show that Axl is expressed on mammary epithelial stem/progenitor cells and regulates multipotent progenitor activity. Thus Axl signal transduction represents a novel regulatory pathway linking normal mammary stem/progenitor cells and breast cancer stem cell activity. Clinical Axl-inhibitors represent a novel therapeutic opportunity to treat aggressive breast cancer.

Citation Format: Crina Tiron, Fanny Pelissier, Katarzyna Wnuk-Lipinska, Ingunn Stefansson, Reeta Virtakoivu, Masaru Miyano, Tone Sandal, David Micklem, James Garbe, Martha Stampfer, Johanna Ivaska, Lars Akslen, Mark LaBarge, James Lorens. Axl signaling is required for stem cell traits and metastasis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C76.
Abstract C8: Bone metastasis seed preselection by the primary tumor stroma
Authors: Zhang Xiang , Jin Xin , Malladi Srinivas , Wen Yong , Brogi Edi , Massague Joan
Published: August 2014

A key question in understanding the origin of metastasis is how cancer cells in a primary tumor acquire the ability to colonize a distant organ. Here we provide one answer to this puzzle by proposing the “metastasis seed pre-selection” model: when the stroma of a primary tumor resembles that of a distant organ, it pre-selects cancer cells that are predisposed to survive in that organ. In breast cancer, the mesenchymal stromal components cancer-associated fibroblasts (CAFs) create a microenvironment similar to that of the bone marrow. Heterogeneous cancer cell populations growing in a CAF-rich breast tumor are skewed towards a preponderance of clones with a superior ability to survive and proliferate on the environmental cues CAFs produce. When these cancer cell clones metastasize and infiltrate the bone marrow, they can maximally exploit these cues familiar to them for initial survival. As a result, these cells have a better chance to colonize the bone when they metastasize. Thus, a CAF-rich stroma primes the breast tumor as a whole at risk of developing bone metastasis. Our findings have implications for the evolution of metastatic traits and the interplay between a primary tumor and its distant metastases.

Citation Format: Xiang Zhang, Xin Jin, Srinivas Malladi, Yong Wen, Edi Brogi, Joan Massague. Bone metastasis seed preselection by the primary tumor stroma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C8.
Abstract C85: FOXM1-dependent gene expression program controls cancer stem cell and metastasis properties of breast cancer cells
Authors: Bergamaschi Anna , Madak-Erdogan Zeynep , Lu Hailing , Katzenellenbogen Benita S.
Published: August 2014

The transcription factor FOXM1 coordinates expression of late cell cycle-related genes. Elevated expression of FOXM1 in breast cancer correlates with a more aggressive tumor phenotype and, as we have previously shown, is associated with resistance to endocrine treatment via 14-3-3ζ protein signaling (Bergamaschi A. et al. Breast Cancer Research 2011, 13:R7). To better understand the role that FOXM1 plays in endocrine resistance and aggressiveness, we analyzed its genome-wide DNA binding and effects on gene expression. We mapped genome-wide FOXM1 binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in breast cancer cell lines after estrogen or tamoxifen treatments. We also investigated the relationship between FOXM1, estrogen receptor-α and MAPK chromatin binding events by clustering analysis of Chip-seq data and identified specific clusters of colocalized binding events as identifiers for different functional gene classes. Clusters enriched in colocalized factor binding sites were associated with genes involved in maintenance of stem cell properties and genes involved in invasion and metastasis (Rho-GTPases). Binding profiles were also integrated with expression data from FOXM1 knockdown experiments, to reveal specific FOXM1 transcriptional networks.

Cell studies showed the relevance of FOXM1 in breast cancer endocrine resistance and development of metastasis. We modulated the levels of FOXM1 and some newly discovered FOXM1 regulated genes in MCF7 cells and assessed their roles in hormone resistance and breast cancer aggressiveness by several functional studies. Increased expression levels of FOXM1 were associated with an increase in the cancer stem cell population. Moreover, FOXM1 overexpressing cell lines exhibited invadopodia which correlated with an increased invasion potential. The use of a specific and selective FOXM1 inhibitor proved to be especially effective in restoring endocrine sensitivity and in decreasing breast cancer aggressiveness. Collectively, our findings define FOXM1 as a master regulator of Rho-GTPase and stem cell marker expression. Dysregulation of the FOXM1 pathway promotes breast cancer progression and therefore makes FOXM1 a relevant therapeutic target to be considered for reducing invasiveness and enhancing response to endocrine and other therapies.

Citation Format: Anna Bergamaschi, Zeynep Madak-Erdogan, Hailing Lu, Benita S. Katzenellenbogen. FOXM1-dependent gene expression program controls cancer stem cell and metastasis properties of breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C85.
Abstract C94: Systemic and stromal support of triple-negative breast cancer progression is prevented by EGF and IGF-1 inhibitors
Authors: Castano Zafira , Marsh Tim , Tadipatri Ramya , Kuznetsov Hanna S. , Al-Shahrour Fatima , Paktinat Mahanaz , Greene-Colozzi April , Nilsson Bjorn , Richardson Andrea L. , McAllister Sandra S.
Published: August 2014

By the time metastatic breast cancer is detected, patients are no longer treated with curative intent. Women with triple-negative breast cancer (TNBC) are particularly at risk for early recurrence. Clinical evidence indicates that in women with metastatic TNBC, the primary tumor and disseminated cells co-existed for an indefinite period of time. We demonstrate that outgrowth of distant tumors that would otherwise remain indolent in cancer-free mice is accelerated in mice with TNBC. Systemic signals provided by TNBCs cause formation of a tumor-supportive microenvironment at distant tumor sites. This microenvironment is enriched for EGF and IGF-1, which convert indolent tumors to a malignant state, defined by expression of factors associated with pluripotency, proliferation, and epithelial-mesenchymal transition. Inhibition of both EGFR and IGF1R maintains these tumors in the indolent state. These results suggest that recurrence rates can be dictated by host systemic factors and offer novel therapeutic potential for TNBC patients.

Citation Format: Zafira Castano, Tim Marsh, Ramya Tadipatri, Hanna S. Kuznetsov, Fatima Al-Shahrour, Mahanaz Paktinat, April Greene-Colozzi, Bjorn Nilsson, Andrea L. Richardson, Sandra S. McAllister. Systemic and stromal support of triple-negative breast cancer progression is prevented by EGF and IGF-1 inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C94.