A high‐performance cell‐labeling NIR‐II dye for in vivo cell tracking

Emerging evidence suggests that the SAM pointed domain containing ETS transcription factor (SPDEF) plays a significant role in tumorigenesis in prostate, breast, colon, and ovarian cancer. However, there are no in vivo studies with respect to the role of SPDEF in tumor metastasis. The present study examined the effects of SPDEF on tumor cell metastasis using prostate tumor cells as a model. Utilizing two experimental metastasis models, we demonstrate that SPDEF inhibits cell migration and invasion in vitro and acts a tumor metastasis suppressor in vivo. Using stable expression of SPDEF in PC3-Luc cells and shRNA-mediated knockdown of SPDEF in LNCaP-Luc cells, we demonstrate for the first time that SPDEF diminished the ability of disseminated tumors cells to survive at secondary sites and establish micrometastases. These effects on tumor metastasis were not a result of the effect of SPDEF on cell growth as SPDEF expression had no effect on cell growth in vitro or subcutaneous tumor xenograft-growth in vivo. Transcriptional analysis of several genes associated with tumor metastasis, invasion, and the epithelial-mesenchymal transition demonstrated that SPDEF expression selectively down-regulated MMP9 and MMP13 in prostate cancer cells. Further analysis indicated that forced MMP9 or MMP13 expression rescued the invasive phenotype in SPDEF expressing PC3 cells in vitro, suggesting that the effects of SPDEF on tumor invasion are mediated, in part, through the suppression of MMP9 and MMP13 expression. These results demonstrate for the first time, in any system, that SPDEF functions as a tumor metastasis suppressor in vivo.

Rho/ROCK signaling and caveolin-1 (Cav1) are implicated in tumor cell migration and metastasis; however, the underlying molecular mechanisms remain poorly defined. Cav1 was found here to be an independent predictor of decreased survival in breast and rectal cancer and significantly associated with the presence of distant metastasis for colon cancer patients. Rho/ROCK signaling promotes tumor cell migration by regulating focal adhesion (FA) dynamics through tyrosine (Y14) phosphorylation of Cav1. Phosphorylated Cav1 is localized to protrusive domains of tumor cells and Cav1 tyrosine phosphorylation is dependent on Src kinase and Rho/ROCK signaling. Increased levels of phosphorylated Cav1 were associated with elevated GTP-RhoA levels in metastatic tumor cells of various tissue origins. Stable expression and knockdown studies of Cav1 in tumor cells showed that phosphorylated Cav1 expression stimulates Rho activation, stabilizes FAK association with FAs, and promotes cell migration and invasion in a ROCK-dependent and Src-dependent manner. Tyrosine-phosphorylated Cav1, therefore, functions as an effector of Rho/ROCK signaling in the regulation of FA turnover and, thereby, tumor cell migration and invasion. These studies define a feedback loop between Rho/ROCK, Src, and phosphorylated Cav1 in tumor cell protrusions, identifying a novel function for Cav1 in tumor metastasis that may contribute to the poor prognosis of some Cav1-expressing tumors.

Ewing sarcoma (ES) is one of the most common and lethal pediatric cancers with a 5-year survival rate of less than 30% for those with metastatic disease. Approximately 25% of ES patients have metastatic disease at time of diagnosis. Current treatment for ES rests on an intensive regimen of five chemotherapeutic agents with a high level of toxicity. Recurrent ES is incurable. Hence, there is a desperate need for more effective and less toxic treatments. A major obstacle in developing new, more effective therapies against ES is the lack of animal models. Zebrafish offers a robust vertebrate model for investigating cancer and evaluating the efficacy of anti-cancer drugs. Many genes, signaling pathways, and mechanisms of carcinogenesis and metastasis are highly conserved between humans and zebrafish. As zebrafish do not develop an adaptive immune response until approximately four weeks of age, they present an ideal system for xenograft tolerance in an immunocompetent organism. In addition, the transparency of zebrafish embryos permits high-resolution optical imaging in vivo and allows for easy tracking of tumor cell migration and metastasis. Our lab has created a ES xenograft model in zebrafish by injecting mCherry-labeled ES cell lines into the transgenic strain Tg(fli:GFP). This strain marks the zebrafish vasculature green, allowing for effective tracking of the injected red cancer cells. Our lab transduced the human ES cell lines A673 and MHH-ES-1, both of which contain the EWSR1:FLI fusion and TP53 mutations, with the mCherry construct. The top 10% brightest cells were selected to allow for easy visualization within the zebrafish. Approximately 200 labeled cells were injected into the yolk at two days post fertilization (2 dpf). Cell growth and migration were tracked until 5 dpf. Using fluorescent microscopy, images of each injected fish were collected at one day post injection (1 dpi), corresponding to 3 dpf, and at 3dpi, corresponding to 5 dpf. Using ImageJ software, these images were carefully analyzed for tumor growth, cell migration, and metastasis. To quantify tumor invasion and metastatic dissemination, we used flow cytometry to compare total ES cell numbers between 1 dpi and 3 dpi within the zebrafish. Our results show that metastasis within the zebrafish, which is indicated by ES cell growth within the tail region, occurs in approximately 15% of injected fish. Data from fluorescent microscopy and flow cytometry demonstrate primary tumor growth, within the zebrafish yolk, occurs in nearly all injected fish. Flow cytometry work is ongoing to quantify the percent increase in tumor growth within injected fish at 5dpf. Large–scale drug screens can be easily performed in the zebrafish due to their small size and ease of drug delivery, which consists simply of immersing fish into drug solutions. Ongoing work includes testing our xenograft model using two known chemotherapeutic agents against ES, doxorubicin and ifosfamide, with the goal of providing a benchmark for novel drugs’ responses on cell migration and metastases. Citation Format: Rebecca A. Anderson, Usua Oyarbide Cuervas-Mon, Seth Corey. Patterns and quantitation of migration and metastasis in a zebrafish xenograft model of ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B007.

Of the five different molecular subtypes of breast cancer, HER2-positive patients have the worst overall prognosis and shortest relapse-free survival. We have shown that HER2-positive breast tumors co-express the oncogenic HER2 isoform, HER2Δ16, which is associated with metastasis and trastuzumab resistance. Here we hypothesize that HER2Δ16 represents the transforming form of HER2 and drives tumorigenesis through deregulation of microRNA (miR) expression. We compared global miR expression profiles between parental MCF-7 breast cancer cells (MCF-7/pcDNA) and a MCF-7 cell line with ectopic expression of HER2Δ16 (MCF-7/HER2Δ16) and found altered regulation of multiple miRs. Interestingly, we observed a 4.8 fold suppression of the tumor suppressor microRNA-7 (miR-7) in the MCF-7/HER2Δ16 cells. MiR-7 has been shown to regulate EGFR expression and clinically, the coexpression of HER2 and EGFR in primary breast tumors is associated with significantly worse prognosis than tumors with HER2 or EGFR expression alone. To determine the impact of miR-7 on EGFR expression and breast tumor cell proliferation and migration, we reestablished miR-7 expression in the highly invasive MCF-7/HER2Δ16 cell line (MCF-7/HER2Δ16/miR-7) and we suppressed miR-7 expression in the non-invasive MCF-7 cell line (MCF-7/miR-7KD). As predicted, reexpression of miR-7 in MCF-7/HER2Δ16 cells dramatically reduced EGFR protein levels, while suppression of miR-7 in MCF-7 cells increased EGFR protein. Furthermore, the reestablished miR-7 expression in MCF-7/HER2Δ16 cells significantly reduced the size and numbers of colonies in a colony formation assay, caused a G1 arrest of the cell cycle and abolished cell migration to the levels observed in noninvasive parental MCF-7 cells. Conversely, inhibition of miR-7 in MCF-7 cells resulted in an increase in colony formation and S-phase of the cell cycle. Taken together, these results indicate that suppression of miR-7 is necessary but not sufficient to promote breast tumor cell migration, while miR-7 directly regulates cellular proliferation. To investigate the contribution of EGFR to HER2Δ16 tumorigenesis, we silenced EGFR expression using shRNAs (MCF-7/HER2Δ16/EGFRKD). While the MCF-7/HER2Δ16/miR-7 cells inhibited colony formation, the MCF-7/HER2Δ16/EGFRKD cells showed enhanced colony formation. However, suppression of EGFR significantly reduced HER2Δ16-induced cell migration activity. These results indicate that EGFR is an essential component of the HER2Δ16 cell migration pathway, but is dispensable for HER2Δ16 colony formation activity. Interestingly, reexpression of miR-7 in the MCF-7/HER2Δ16 cell line resulted in a loss of Src kinase phosphorylated activation. Our preliminary results suggest that Src kinase activation in the absence of miR-7 drives HER2Δ16 tumor cell proliferation. We are currently investigating the contribution of miR-7 to HER2Δ16-induced resistance to both trastuzumab and lapatinib. In summary, HER2Δ16 represents a novel breast oncogene that promotes breast tumor cell proliferation and metastasis through suppression of miR-7 and cooperation with Src kinase. Our data suggests that modulation of miR-7 expression can be implemented as a therapeutic approach for the treatment of the most aggressive and therapeutically refractory HER2-positive metastatic breast tumors. Citation Format: Felicia C. Huynh, Frank E. Jones. HER2Δ16, the oncogenic isoform of HER2, suppresses microRNA-7 to promote breast tumor cell migration and proliferation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A008.

GPR4, a G protein-coupled receptor, has been shown to function as a proton sensor. Within tumor microenvironments, the interstitial acidic pH may activate GPR4 to regulate the behavior of tumor cells, blood vessels, and other cells. The expression of GPR4 is detected in some tumor cells, and it is the focus of this study to investigate the effects of acidosis/GPR4 signaling on tumor cell migration and metastasis. B16F10 melanoma cells and TRAMP-C1 prostate cancer cells, genetically engineered to overexpress GPR4 or the control vector, were subject to a series of cell motility assays. Transwell and wound closure assays were performed to examine cell migration, and it was observed that upon GPR4 activation in an acidic pH, cell migration was inhibited two to four fold in comparison to the vector control. These same trends were observed for invasion assays, in which Matrigel or a HUVEC endothelial monolayer was applied to the top of each transwell filter. To elucidate the molecular mechanism by which acidosis/GPR4 mediated motility inhibition occurs, G-LISA RhoA activation assays were used and it was observed that upon GPR4 activation in an acidic environment, the levels of activated RhoA, when compared to the vector control, were increased two and three fold. Furthermore, activation of Rho by its activator CN01 in B16F10 and TRAMP-C1 tumor cells recapitulated the acidosis/GPR4 induced motility inhibition phenotype, while inhibition of Rho reversed such phenotype. To examine the biological effects in vivo, B16F10 cells were injected into C57BL/6 female mice through tail veins. Pulmonary melanoma metastasis was inhibited by greater than 80% in GPR4 overexpressing B16F10 cells in comparison to the vector control. Upon treatment with the Rho activator CN01 in culture, the phenotype of the B16F10 vector cells paralleled that of the GPR4 overexpressing cells as metastasis was inhibited by more than 90%. These findings suggest that GPR4 activation by acidic pH plays a role in the inhibition of migration and invasion in B16F10 and TRAMP-C1 tumor cells, and the activation of Rho is at least partly responsible for this phenotype on the molecular level. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5278.

Tumor metastasis is responsible for ~90% of all cancer deaths. One of the key steps of tumor metastasis is tumor cell migration and invasion. Filopodia are cell surface extensions that are critical for tumor cell migration. Fascin protein is the main actin-bundling protein in filopodia. Small-molecule fascin inhibitors block tumor cell migration, invasion, and metastasis. Here we present the structural basis for the mechanism of action of these small-molecule fascin inhibitors. X-ray crystal structural analysis of a complex of fascin and a fascin inhibitor shows that binding of the fascin inhibitor to the hydrophobic cleft between the domains 1 and 2 of fascin induces a ~35o rotation of domain 1, leading to the distortion of both the actin-binding sites 1 and 2 on fascin. Furthermore, the crystal structures of an inhibitor alone indicate that the conformations of the small-molecule inhibitors are dynamic. Mutations of the inhibitor-interacting residues decrease the sensitivity of fascin to the inhibitors. Our studies provide structural insights into the molecular mechanism of fascin protein function as well as the action of small-molecule fascin inhibitors.

Chronic inflammation is associated with cancer. CXCL8 promotes tumor microenvironment construction through recruiting leukocytes and endothelial progenitor cells that are involved in angiogenesis. It also enhances tumor cell proliferation and migration. Metformin, type II diabetes medication, demonstrates anticancer properties via suppressing inflammation, tumor cell proliferation, angiogenesis, and metastasis. This study intended to address the role of metformin in regulation of CXCL8 expression and cell proliferation and migration. Our data indicated that metformin suppressed LPS-induced CXCL8 expression in a dose-dependent manner through inhibiting NF-κB, but not AP-1 and C/EBP, activities under the conditions we used. This inhibitory effect of metformin is achieved through dampening LPS-induced NF-κB nuclear translocation. Cell migration was inhibited by metformin under high dose (10 mM), but not cell proliferation.

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.

CD44, a transmembrane receptor for hyaluronic acid, is implicated in various adhesion-dependent cellular processes, including cell migration, tumor cell metastasis and invasion. Recent studies demonstrated that CD44 expressed in cancer cells can be proteolytically cleaved at the ectodomain by membrane type 1-matrix metalloproteinase (MT1-MMP) to form soluble CD44 and that CD44 cleavage plays a critical role in cancer cell migration. Here, we show that transforming growth factor-beta (TGF-beta), a multifunctional cytokine involved in cell proliferation, differentiation, migration and pathological processes, induces MT1-MMP expression in MDA-MB-435s cells. TGF-beta-induced MT1-MMP expression was blocked by the specific extracellular regulated kinase-1/2 (ERK1/2) inhibitor PD98059 and the specific phosphoinositide 3-OH kinase (PI3K) inhibitor LY294002. In addition, treatment with SP600125, an inhibitor for c-Jun NH(2)-terminal kinase (JNK), resulted in a significant inhibition of MT1-MMP production. These data suggest that ERK1/2, PI3K, and JNK likely play a role in TGF-beta-induced MT1-MMP expression. Interestingly, treatment of MDA-MB-435s cells with TGF-beta resulted in a colocalization of MT1-MMP and CD44 in the cell membrane and in an increased level of soluble CD44. Using an electric cell-substrate impedance sensing cell-electrode system, we demonstrated that TGF-beta treatment promotes MDA-MB-435s cell migration, involving MT1-MMP-mediated CD44 cleavage. MT1-MMP siRNA transfection-inhibited TGF-beta-induced cancer cell transendothelial migration. Thus, this study contributes to our understanding of molecular mechanisms that play a critical role in tumor cell invasion and metastasis.

STAT2 is a pivotal signaling component in the antiviral and antiproliferative effects of type I and type III interferon (IFN), but its role in cancer remains understudied. To date, only IFNs are known to activate STAT2. Work by us and others has demonstrated that STAT2 deficiency impairs the antitumor effects of type I IFN, supporting its role in suppressing tumor growth. With the availability of Stat2-/- mice, we interrogated the tumor-suppressive role of STAT2 by employing the AOM/DSS model of colitis-associated cancer and the spontaneous ApcMin/+ model of colorectal cancer (CRC). In both cancer models, loss of STAT2 reduced the number of adenomas, showing that under specific conditions STAT2 can be tumorigenic. Next, immunohistochemical staining of human normal colon and CRC tumors revealed elevated STAT2 protein in both adenomas and adenocarcinomas, suggesting that STAT2 is implicated in human CRC. Conversion of adenomas to adenocarcinomas is linked to loss of p53 function, which occurs in >50% of colorectal cancers. Therefore, we evaluated whether STAT2 altered the aggressive phenotype of p53-deficient colon carcinoma cells. We silenced STAT2 in isogenic p53 null and p53 wild-type tumor cells. STAT2 knockdown in p53 null colon carcinoma cells reduced migration of 3D tumor spheroids and tumor colony formation while no effect was seen in p53 wild-type tumor cells. We also observed that STAT2 knockdown in p53 null tumor cells increased protein levels of the marker of epithelial-mesenchymal transition, E-cadherin. Conversely, overexpression of STAT2 in p53 null cells reduced E-cadherin protein. In vivo, STAT2 knockdown delayed subcutaneous tumor growth of p53 null tumor cells. Furthermore, using an experimental liver metastasis tumor model, STAT2 knockdown abrogated the formation of tumor nodules in the liver. RNA-seq profiling of subcutaneous p53 null tumors revealed a subset of genes that were differentially expressed in a STAT2-dependent manner. Clinically, analysis of the TCGA database showed a STAT2 transcriptional signature in colorectal tumors that we are presently correlating with p53 alterations. Overall, we conclude that STAT2 enhances tumor growth and can contribute to disease progression by promoting migration and invasion when normal p53 function is lost. Citation Format: Ana M. Gamero, Kevin Kotredes, Aliza Abezis, Sruthi R. Gohimukkula, Alexandra Afanassiev, Tess Cremers, John J. Swain. p53 inactivation and STAT2 cooperate to enhance migration and metastasis of colon tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3157.

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2alpha (AP-2alpha) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2alpha binding elements, AP-2alpha-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2alpha into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2alpha wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2alpha regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2alpha directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2alpha and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2alpha in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.

Introduction: Breast cancer is a heterogeneous disease, and is comprised of a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we targeted DCLK1 as a novel therapeutic strategy in breast cancer cells to reduce cancer cell self-renewal, tumorigenesis and metastasis. Methods: Anti-DCLK1-siRNA was transfected into breast cancer cell lines (MCF7 [luminal] and MDAMB231 [basal-triple negative breast cancer cell line]). For analysis of circulating tumor cells, MDAMB231/MCF7 cells were stably transfected with RFP (red fluorescence protein). Tumor cell colony formation, mammosphere formation (self-renewal), cell migration and invasion were assessed to evaluate the effect of DCLK1 knockdown and or DCLK1 overexpression on breast cancer cells self-renewal and metastatic potential. Xenograft model was used to identify the role of DCLK1 on breast cancer cell growth and circulating tumor cells in vivo. The data were generated utilizing experimental protocols for clonogenic culture, circulating tumor cells, immunohistochemistry, RT-qPCR and Western blotting. Results: Expression of DCLK1 was ∼36% higher in human breast cancer tissues than in normal breast tissue. Silencing DCLK1 via RNA interference decreased the colony formation (∼70%), and self-renewal ability (∼85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to less (40-80%) breast cancer cells migration and invasion. Furthermore, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were markedly reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in xenograft tumors resulted in reduced tumor growth (∼81%) and CTCs (∼95%) in vivo. However, overexpression of DCLK1 in the non-metastatic MCF7 breast cancer cell line increased its in vitro migration and invasion potency and pluripotency along with in vivo tumor formation and progression. Conclusions: DCLK1 expression is associated with breast cancer cell self-renewal, metastasis and tumorigenesis. Targeting DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis and tumor growth. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer. Citation Format: PARTHASARATHY CHANDRAKESAN, Nathaniel Weygant, Dongfeng Qu, William Berry, Randal May, Naushad Ali, Sripathi Sureban, Eddie Bannerman-Menson, Michael Schlosser, Courtney Houchen. Knocking down the expression of DCLK1 reduces mammary tumor formation, tumor cell self-renewal and metastasis: DCLK1 a novel therapeutic target 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 2493.

Polysialic acid (polySia) is a linear alpha-2,8-linked carbohydrate homopolymer of up to 100 sialic acid residues. It is characteristically re-expressed on the surface of NCAM (neuronal cell adhesion molecule) in many cancer cells where it modulates tumour dissemination. PolySia-NCAM expression is strongly associated with poor clinical prognosis and correlates with aggressive/invasive disease in small cell lung cancer, pancreatic cancer, neuroblastoma and many other tumours principally of neural crest origin [1]. SiRNA knockdown of polysialyltransferases (polySTs) ST8SiaII and ST8SiaIV, the enzymes responsible for polysialylation of neural cell adhesion molecule (NCAM), has been shown to abolish cell migration in tumour cells [2]. Besides brain regions with persistent neuronal plasticity, polySia is essentially absent from the adult body. Its exclusive synthesis by two polySTs, ST8SiaIV and particularly ST8SiaII, present a novel, selective, highly attractive but largely unexplored therapeutic opportunity [1]. We have embarked on a programme of design and synthesis of novel polyST inhibitors, and have established a screening cascade to determine enzyme inhibition and effects on polySia-expressing cells in vitro. Using endoneuraminidase, an enzyme that specifically cleaves polySia chains from NCAM, we can assess the rate and extent to which polySia growth is inhibited in the presence of agent in cellular systems. In addition, an in vitro scratch assay was established to evaluate modulation of cell migration using a transfected isogenic cell line system (C6-STX: polySia +, ST8SiaII + / C6-WT: polySia -, ST8SiaII - [3]) and neuroblastoma cell lines known to express polySia-NCAM, ST8SiaIV and ST8SiaII (SHSY-5Y and IMR-32). We have identified compounds, including ICT-3067, ICT-3128 and ICT-3147, which lead to a significant reduction in cell surface polySia-NCAM and that modulate tumour cell migration in in vitro. We have also identified a number of key modifications to CMP-sialic acid precursor molecules which dramatically decrease cell migration in ST8SiaII-expressing cells, through modulation of polySia assembly. It is also noteworthy that the potency of these compounds has been increased with simple chemical modifications, resulting in greater lipophilicity. In summary, a number of efficient modulators of polySia assembly and their efficacy in reducing cell migration is described. This points to the potential of the polySia biosynthesis pathway, and in particular the polySTs, as attractive therapeutic targets in metastatic tumours. [1] Falconer, R.A. et al., Curr. Cancer Drug Targets, 2012, 12, 925-939; [2] Schreiber et al., Gastroenterology, 2008, 134, 1555-1566; [3] Suzuki, M. et al., Glycobiology, 2005, 15, 887-894. Citation Format: Bradley R. Springett, Yousef M.J. Al-Saraireh, Virginie Viprey, Mark Sutherland, Melanie Begouin, Matthew Northrop, Rida Saeed, Paul M. Loadman, Laurence H. Patterson, Steven D. Shnyder, Robert A. Falconer. Inhibition of cell surface polysialic acid biosynthesis modulates tumor cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4410. doi:10.1158/1538-7445.AM2013-4410

Despite the acknowledged impact of the tumor stroma on breast cancer development and progression, the molecular basis of such effects remain partially unexplained. We previously reported that breast cancer-associated fibroblasts (CAFs) induced epithelial-mesenchymal transition and an increase in cell membrane fluidity and migration speed in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. More recently, in order to better define the mechanisms responsible for the CAF-promoted tumor cell migration, we investigated the role of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating membrane fluidity, and demonstrated its CAF-triggered up-regulation as well as its crucial role in the migratory ability of the above tumor cells. Besides SCD1 induction, a CAF-promoted enhancement in the protein level and/or activity of the SCD1 transcription factor, the sterol regulatory element-binding protein 1 (SREBP1), was observed. Moreover, the influence of stroma-derived signals in cancer cell migration speed was proved by cell tracking analysis in the presence of neutralizing antibodies to hepatocyte growth factor, transforming growth factor-β or basic fibroblast growth factor, where a marked reduction or abolishment of the fibroblast-triggered increase in cancer cell migration speed was observed. In the last part of this study, in order to verify if soluble CAF-derived factors stimulate breast cancer cell migration in a SCD1-dependent manner, tumor cells were exposed to CAF-conditioned medium (CM) and their migration evaluated by scratch assay in the presence of a small molecule inhibitor of SCD1. Moreover, to assess if the induction of SCD1 expression by CAFs might occur via SREBP1, the desaturase levels were also determined in SREBP1-inhibited tumor cells. These latest investigations indicate that SCD1 contributes to the promotion of breast cancer cell migration by CAF-derived soluble factors, since the desaturase inhibition completely suppressed the stimulatory effect of CAF-CM on tumor cell migration. SREBP1 inhibition impaired CAF-mediated up-regulation of SCD1 in poorly invasive but not in highly invasive tumor cells, in which SREBP1-independent mechanisms may account for the enhancement of SCD1 levels. These results provide further insights in understanding the role of CAFs in promoting tumor cell migration, which may help to design new stroma-based therapeutic strategies.

Cell migration plays an important role in many physiological and pathological processes, including tumor metastasis. Tumor cell migration is increased through the sequential induction of HIF-1α and VEGF under hypoxic conditions. Thymosin β-4 (Tβ4) is an actin-sequestering protein which controls cytoskeletal reorganization. Here, we investigated whether tumor cell migration could be co-operatively controlled by hypoxia and Tβ4. Cell migration was measured by wound healing assay with scratching confluent monolayers of tumor cells. Cell migration was enhanced 18 h after scratching cells. In addition, we found that the expression of HIF-1α, VEGF-A isoform 164/120 and Tβ4 was increased by scratching cells. Cell migration was decreased by the inhibition of Tβ4 or HIF-1α expression with lentiviral shRNA of Tβ4 or siRNA of HIF-1α, respectively. In contrast, cell migration was increased by the treatment with Tβ4 proteins. The inhibitory effect of Tβ4-shRNA or HIF-1α-siRNA was also attenuated by treatment with Tβ4 proteins. Collectively, these findings suggest that Tβ4 and HIF-1α cooperatively enhance tumor cell migration.