Tumor Metastatic Cascade Research Articles

Platelet-hitchhiking biomimetic nanoplatforms pry precisely tumor seed and growing soil for inhibiting tumor recurrence and metastasis

The theory of “seed and soil” illustrated that the tumor microenvironment and circulating tumor cells (CTCs) facilitated the tumor relapse and metastasis. An effective strategy should synergetically eliminate the seeds cells and reshape the tumor microenvironment. Herein, a platelet-hitchhiking biomimetic nanoplatform was constructed to pry precisely tumor seed and growing soil for inhibiting tumor recurrence and metastasis via “platelet bridge”. Benefiting from the PSN peptides, the nanoplatform captured and eliminated CTCs accurately in blood by specifically binding between PSN peptide and surface P-selectin on activated platelets, thus inhibiting hematogenous metastasis. Attributing to precise delivery from tumor targeting of the cell membrane and fluorescence imaging guidance of new indocyanine green, the nanoplatform accumulated at primary tumor. Triggered by the external laser and acidic tumor microenvironment, it degraded and robustly released doxorubicin and zinc ions. Along with the onsite photothermal combined chemotherapy, the excessive zinc ions disturbed the energy metabolism for triggering immune response. They together regulated immune suppressive microenvironment to prevent tumor invasion and metastasis. The platelet-hitchhiking biomimetic nanoplatform interfered the “seed and soil” crosstalk to achieve a two-pronged approach of eliminating the CTCs and reshaping tumor microenvironment, which synergistically inhibited multiple pathways in the tumor recurrent and metastasis cascade.

Abstract IA017: The polyaneuploid cancer cell state as a mediator of therapeutic resistance

Abstract More than ten million people around the world will die from metastatic cancer in 2022. To date, no common genomic signature has been defined that can explain or identify which patients will develop lethal metastatic cancer. In all of these incurable patients lethal cancer independently evolves capacities for metastasis, avoidance of all cellular death pathways, and resistance. This convergent outcome demands an explanation beyond the slow and steady accrual of stochastic mutations. Generally, it is thought that cancer cells utilize the forces of natural selection when utilizing accumulating mutations during its evolution. Their utilization of heritable variation permits the cancer cell population to respond and survive environmental hazards. The tumor ecosystem is exposed to extreme and constantly changing chemical conditions. These changes can be natural, (e.g., loss of blood supply) or imposed (therapeutic). While the nature of these catastrophes may be varied or unique, their common property may be to doom the current cancer phenotype unless it evolves rapidly. We propose a complementary theory to explain cancer cell fitness through these environmental hazards that lead to metastasis, therapeutic resistance, and lethality. The basis of this theory is that a subset of cancer cells accesses evolutionary or developmental (evo/devo) stress survival programs that enable whole genome doubling (polyploidization) and cell cycle arrest. These stress survival programs are observed across the tree of life, from unicellular both prokaryotes and eukaryotes, such as Archaea, Bacteria, and yeast, to multicellular organism including plants and animals. In cancer, environmental stress and these stress programs result in cancer cell survival though a state of cell cycle arrest that is simultaneously polyploid and aneuploid. During this polyaneuploid cancer cell (PACC) state, cancer cells pause their cell cycle and protect the integrity of their DNA. This avoids the environmental stresses associated with the tumor microenvironment and metastatic cascade as well as the toxic effects of systemic therapy. In addition, by doubling their DNA and increasing their RNA and protein material, cancer cells in the PACC state have increased potential to develop further resistance mechanism to environmental stressors and otherwise toxic therapies. We have demonstrated that upon removal of stress, e.g., chemotherapy, cells in the PACC state undergo depolyploidization and generate resistant progeny. Thus, cells in the PACC state may play a critical role in lethality by their increased capacity to endure novel and stressful environments. This enables the metastatic phenotype and the generation of heritable variation that can be dispensed to their 2N+ aneuploid progeny, providing population rescue in response to therapeutic stress. Targeting cells in the PACC state is essential to cancer therapy and patient cure. Citation Format: Kenneth J. Pienta. The polyaneuploid cancer cell state as a mediator of therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA017.

The Role of Innate Immune Cells in Tumor Invasion and Metastasis.

Simple SummaryTumor invasion and metastasis are one of the main reasons patients succumb to cancer. In this review, we summarize recent studies which provide evidence on the involvement of cells of the innate immune system and their function in invasion and metastasis.Metastasis is considered one of the hallmarks of cancer and enhanced tumor invasion and metastasis is significantly associated with cancer mortality. Metastasis occurs via a series of integrated processes involving tumor cells and the tumor microenvironment. The innate immune components of the microenvironment have been shown to engage with tumor cells and not only regulate their proliferation and survival, but also modulate the surrounding environment to enable cancer progression. In the era of immune therapies, it is critical to understand how different innate immune cell populations are involved in this process. This review summarizes recent literature describing the roles of innate immune cells during the tumor metastatic cascade.

Store-operated Ca2+ entry as a key oncogenic Ca2+ signaling driving tumor invasion-metastasis cascade and its translational potential.

Tumor metastasis is the primary cause of treatment failure and cancer-related deaths. Store-operated Ca2+ entry (SOCE), which is mediated by stromal interaction molecules (STIM) and ORAI proteins, has been implicated in the tumor invasion-metastasis cascade. Epithelial-mesenchymal transition (EMT) is a cellular program that enables tumor cells to acquire the capacities needed for migration and invasion and the formation of distal metastases. Tumor-associated angiogenesis contributes to metastasis because aberrantly developed vessels offer a path for tumor cell dissemination as well as supply sufficient nutrients for the metastatic colony to develop into metastasis. Recently, increasing evidence has indicated that SOCE alterations actively participate in the multi-step process of tumor metastasis. In addition, the dysregulated expression of STIM/ORAI has been reported to be a predictor of poor prognosis. Herein, we review the latest advances about the critical role of SOCE in the tumor metastasis cascade and the underlying regulatory mechanisms. We emphasize the contributions of SOCE to the EMT program, tumor cell migration and invasion, and angiogenesis. We further discuss the possibility of modulating SOCE or intervening in the downstream signaling pathways as a feasible targeting therapy for cancer treatment.

Neutrophil Extracellular Traps in Tumor Metastasis: Pathological Functions and Clinical Applications.

Simple SummaryTumor-associated neutrophils constitute an important portion of the infiltrating immune cells in the tumor microenvironment. One of the abilities of neutrophils is forming neutrophil extracellular traps. Recent studies on tumor-associated neutrophils have drawn increasing attention to the role of neutrophil extracellular traps in the tumor microenvironment. There were also some reviews summarize the pro-tumorigenic activity of NETs in tumors. The specific novelty of this article is the specific summarization on the pivotal roles of NETs in tumor invasion-metastasis cascade and the recapitulation on the potential of NETs in clinical applications. Neutrophil extracellular trap (NET) formation is an ability of neutrophils to capture and kill pathogens by releasing chromatin scaffolds, along with associated cytotoxic enzymes and proteases, into the extracellular space. NETs are usually stimulated by pathogenic microorganisms and their products, surgical pressure or hypoxia. Interestingly, a number of recent studies suggest that tumor cells can induce NET formation, which in turn confers tumor cell malignancy. Notably, emerging studies indicate that NETs are involved in enhancing local invasion, increasing vascular permeability and facilitating immune escape and colonization, thus promoting tumor metastasis. In this article, we review the pivotal roles of NETs in the tumor metastasis cascade. We also recapitulate the potential of NETs as a cancer prognostic biomarker and therapeutic target.

Reprogramming the tumor metastasis cascade by targeting galectin-driven networks.

A sequence of interconnected events known as the metastatic cascade promotes tumor progression by regulating cellular and molecular interactions between tumor, stromal, endothelial, and immune cells both locally and systemically. Recently, a new concept has emerged to better describe this process by defining four attributes that metastatic cells should undergo. Every individual hallmark represents a unique trait of a metastatic cell that impacts directly in the outcome of the metastasis process. These critical features, known as the hallmarks of metastasis, include motility and invasion, modulation of the microenvironment, cell plasticity and colonization. They are hierarchically regulated at different levels by several factors, including galectins, a highly conserved family of β-galactoside-binding proteins abundantly expressed in tumor microenvironments and sites of metastasis. In this review, we discuss the role of galectins in modulating each hallmark of metastasis, highlighting novel therapeutic opportunities for treating the metastatic disease.

MiR-361-3p regulates ERK1/2-induced EMT via DUSP2 mRNA degradation in pancreatic ductal adenocarcinoma

Metastasis remains one of the most intractable challenges in pancreatic ductal adenocarcinoma (PDAC) biology, and epithelial-to-mesenchymal transition (EMT) is essential to the epithelium-originated solid tumor metastasis cascade. Emerging evidence demonstrates that aberrant miRNA expression is involved in pancreatic cancer progression. We found that miR-361-3p was associated with an advanced stage of PDAC and poor prognosis. Hence, the effect of miR-361-3p on metastasis of PDAC cells was evaluated using Transwell assay and wound healing assay in vitro as well as orthotopic and liver metastasis pancreatic cancer models in vivo. Overexpression of miR-361-3p promoted pancreatic cancer cell migration and invasion in vitro, and miR-361-3p-elevated PDAC cells were prone to generating metastatic nodules in vivo. However, miR-361-3p showed no significant effect on the proliferation of PDAC cells in vivo or in vitro. Further study demonstrated that miR-361-3p could enhance EMT and ERK pathway activation, and ERK inhibitor could attenuate miR-361-3p-induced EMT. Luciferase assays, qPCR, and western blot and Ago2 co-immunoprecipitation were performed to identify the direct target of miR-361-3p. Mechanistic investigations identified DUSP2 as a direct target of miR-361-3p, and DUSP2 was revealed to be involved in miR-361-3p-induced EMT by directly leading to the inactivation of the ERK pathway. Moreover, we found that miR-361-3p-induced EMT was dependent on Ago2, the core component of RNA-induced silencing complex, while enforced expression of Ago2 enhanced the miR-361-3p-induced effect by promoting interference efficacy and specificity rather than regulating miR-361-3p stability and biogenesis. Thus, this study revealed that miR-361-3p functions as an oncomiR for promoting metastasis and identified the miR-361-3p/DUSP2/ERK axis as a novel EMT axis dependent on Ago2 in PDAC.

Osteopontin facilitates tumor metastasis by regulating epithelial-mesenchymal plasticity.

Tumor metastasis leads to high mortality; therefore, understanding the mechanisms that underlie tumor metastasis is crucial. Generally seen as a secretory protein, osteopontin (OPN) is involved in multifarious pathophysiological events. Here, we present a novel pro-metastatic role of OPN during metastatic colonization. Unlike secretory OPN (sOPN), which triggers the epithelial–mesenchymal transition (EMT) to initiate cancer metastasis, intracellular/nuclear OPN (iOPN) induces the mesenchymal–epithelial transition (MET) to facilitate the formation of metastases. Nuclear OPN is found to interact with HIF2α and impact the subsequent AKT1/miR-429/ZEB cascade. In vivo assays confirm that the progression of metastatic colonization is accompanied by the nuclear accumulation of OPN and the MET process. Furthermore, evidence of nuclear OPN in the lung metastases is exhibited in clinical specimens. Finally, VEGF in the microenvironment was shown to induce the translocation of OPN into the nucleus through a KDR/PLCγ/PKC-dependent pathway. Taken together, our results describe the pleiotropic roles of OPN in the tumor metastasis cascade, which indicate its potential as an effective target for both early and advanced tumors.

Hedgehog signaling: modulation of cancer properies and tumor mircroenvironment.

Cancer poses a serious health problem in society and is increasingly surpassing cardiovascular disease as the leading cause of mortality in the United States. Current therapeutic strategies for cancer are extreme and harsh to patients and often have limited success; the danger of cancer is intensified as it metastasizes to secondary locations such as lung, bone, and liver, posing a dire threat to patient treatment and survival. Hedgehog signaling is an important pathway for normal development. Initially identified in Drosophila, the vertebrate and mammalian equivalent of the pathway has been studied extensively for its role in cancer development and progression. As this pathway regulates key target genes involved in development, its action also allows for the modulation of the microenvironment to prepare a tumor-suitable niche by manipulating tumor cell growth, differentiation, and immune regulation, thus creating an enabling environment for progression and metastasis. In this review, we will summarize recent scientific discoveries reporting the impact of the Hedgehog signaling pathway on the tumor initiation process and metastatic cascade, shedding light on the ability of the tumor to take over a mechanism crucially intended for development and normal function.

An evidence-based knowledgebase of metastasis suppressors to identify key pathways relevant to cancer metastasis.

Metastasis suppressor genes (MS genes) are genes that play important roles in inhibiting the process of cancer metastasis without preventing growth of the primary tumor. Identification of these genes and understanding their functions are critical for investigation of cancer metastasis. Recent studies on cancer metastasis have identified many new susceptibility MS genes. However, the comprehensive illustration of diverse cellular processes regulated by metastasis suppressors during the metastasis cascade is lacking. Thus, the relationship between MS genes and cancer risk is still unclear. To unveil the cellular complexity of MS genes, we have constructed MSGene (http://MSGene.bioinfo-minzhao.org/), the first literature-based gene resource for exploring human MS genes. In total, we manually curated 194 experimentally verified MS genes and mapped to 1448 homologous genes from 17 model species. Follow-up functional analyses associated 194 human MS genes with epithelium/tissue morphogenesis and epithelia cell proliferation. In addition, pathway analysis highlights the prominent role of MS genes in activation of platelets and coagulation system in tumor metastatic cascade. Moreover, global mutation pattern of MS genes across multiple cancers may reveal common cancer metastasis mechanisms. All these results illustrate the importance of MSGene to our understanding on cell development and cancer metastasis.

The ectopic expression of Snail in MDBK cells does not induce epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail-induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the down-regulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells.

Reversibility of the Snail-induced epithelial–mesenchymal transition revealed by the Cre–loxP system

The epithelial–mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell–cell junctions and cell polarity, as well as the acquisition of migratory and invasive properties. Snail is an EMT-inducer whose expression in several different epithelial cells, e.g., Madin–Darby canine kidney (MDCK), leads to EMT. To further understand EMT induced by Snail expression, the Cre–loxP site-specific recombination system was used to investigate its reversibility. Transfection of MDCK cells with loxP-flanked Snail (Snail-loxP) resulted in EMT induction, which included the acquisition of a spindle-shaped fibroblastic morphology, the downregulation of epithelial markers, and the upregulation of mesenchymal markers. DNA methylation of the E-cadherin promoter, which often occurs during E-cadherin downregulation, was not observed in Snail+ cells. After Cre-mediated excision of Snail-loxP, the cells reacquired an epithelial morphology, upregulated epithelial markers, and downregulated mesenchymal markers. Thus, EMT induced by Snail expression was reversible.

Expression of focal adhesion kinase in uveal melanoma and the effects of Hsp90 inhibition by 17-AAG

IntroductionFocal adhesion kinase (FAK) is implicated in tumor progression and metastatic cascade, and has been shown to be overexpressed in a variety of human cancers. However, the role of FAK in human uveal melanoma (UM) is not well defined. The purpose of this study was to evaluate the expression of FAK in UM tumors and normal eyes, and to determine the effect of Hsp90 inhibition on FAK expression in UM cells. MethodsFAK expression was assessed in 39 UM specimens, FAK[pY397] expression was assessed in 51 UM specimens, and both FAK and FAK[pY397] expression were assessed in 20 normal eyes. The expression of FAK and FAK[pY397] was detected by Western blot in five UM cell lines after treatment with 10μmol/L of 17-AAG. ResultsFAK was positive in 87.2% and FAK[pY397] in 90% of UM specimens. Low FAK expression was detected in non-tumor structures and in normal eyes. The cell lines with the most proliferative, invasive phenotype (92.1, SP6.5 and MKT-BR) displayed high expression of FAK[pY397], and the levels of FAK and FAK[pY397] were decreased in the presence of 17-AAG starting with 24h of exposure. ConclusionFAK and FAK[pY397] were overexpressed in human UM tumors compared to normal ocular tissue and high levels of FAK[pY397] were seen in the most aggressive UM cell lines. Hsp90 inhibition led to downregulation of FAK expression. We propose a role for FAK in the pathogenesis of UM. Future studies are needed to explore the use of Hsp90 inhibitors as a feasible approach for modulating FAK in UM.

Critical role of miR-10b in transforming growth factor-β1-induced epithelial–mesenchymal transition in breast cancer

Epithelial-mesenchymal transition (EMT) is a key process in the tumor metastatic cascade that is characterized by the loss of cell-cell junctions and cell polarity, resulting in the acquisition of migratory and invasive properties. Recent evidence showed that altered microRNA-10b (miR-10b) expression was implicated in the occurrence of EMT of breast cancer. However, the exact role and underlying mechanisms of miR-10b in the EMT of breast cancer still remain unknown. In this study, miR-10b was found to be upregulated in breast cancer tissues and breast cancer cell lines and the expression of miR-10b was shown to be closely correlated with aggressiveness in breast cancer. Treating breast cancer cells with the miR-10b inhibitor increased E-cadherin expression while decreasing vimentin expression. At the same time, on inhibition of miR-10b, the invasion and proliferation ability of breast cancer cells also decreased. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that induces EMT in multiple cell types. Here, we identified miR-10b as a target gene of TGF-β1. The expression of miR-10b increased during TGF-β1-induced EMT of breast cancer cells. Further study showed that inhibition of miR-10b expression partially reversed the EMT, invasion and proliferation induced by TGF-β1 in breast cancer cells. Taken together, these results demonstrated a novel function for miR-10b in TGF-β1-induced EMT in breast cancer and increased their metastatic potential. MiR-10b might become a possible target for gene therapy in breast cancer.

MicroRNAs and breast cancer

MicroRNAs (miRNAs) are a class of small molecule non-coding RNAs.Those RNAs are considered as oncogenes and tumor suppressor genes in breast cancer and can be a promoter or inhibitor in tumor invasion and metastasis cascade stages,and they are also closely related to the tumor epithelial-mesenchymal transition and tumor stem cell generation.New researches show that miRNAs can serve as significant diagnostic and prognostic indicators to provide a theoretical basis for the biotherapy of targeting miRNAs which is applied to the clinical diagnosis and treatment of breast cancer. Key words: MicroRNAs; Breast neoplasms; Neoplasm metastasis; Diagnosis ; Molecular targeted therapy

The transcription factor LEF-1 induces an epithelial–mesenchymal transition in MDCK cells independent of β-catenin

The epithelial–mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell–cell junctions and cell polarity, as well as the acquisition of migratory and invasive properties. LEF-1 is a member of the lymphoid enhancer-binding factor/T-cell factor (LEF/TCF) family of DNA-binding transcription factors, which interact with nuclear β-catenin and act as central transcriptional mediators of Wnt signaling. To investigate the role of LEF-1 in EMT, we generated stable LEF-1 transfectants using MDCK cells. The transfectants had a spindle-shaped mesenchymal morphology, and enhanced migration and invasiveness relative to control cells. These EMT changes were accompanied by the downregulation of an epithelial marker protein, E-cadherin, and the upregulation of mesenchymal marker proteins, vimentin and N-cadherin. Consistent with these observations, the mRNA levels of Slug, ZEB1, and ZEB2—EMT-related transcription factors—increased significantly. Although the N-terminally deleted mutant LEF-1 cannot interact with β-catenin, it retained the ability to induce EMT. Consistent with these observations, neither the expression of a dominant negative β-catenin/engrailed chimera, nor the expression of a cytoplasmic domain of E-cadherin that sequesters β-catenin from binding to LEF/TCF, reversed LEF-1-induced EMT. Together, these data indicated that the nuclear function of β-catenin was not necessary for the induction of Slug, ZEB1, and ZEB2 expression leading to EMT.

The transcription factor Snail enhanced the degradation of E-cadherin and desmoglein 2 in oral squamous cell carcinoma cells

Epithelial–mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell–cell junctions and cell polarity as well as the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail is a regulator of EMT that represses E-cadherin transcription through its interaction with proximal E-boxes in the promoter region of target genes. To investigate the role of Snail in EMT, we generated stable Snail transfectants using the oral squamous cell carcinoma cell line HSC-4 (Snail/HSC-4). Snail/HSC-4 cells had a spindle-shaped mesenchymal morphology, and enhanced migration and invasiveness relative to control cells. Consistent with these EMT changes, the downregulation of epithelial marker proteins, E-cadherin and desmoglein 2, and the upregulation of mesenchymal marker proteins, vimentin and N-cadherin were detected. Despite these observations, the mRNA levels of E-cadherin and desmoglein 2 did not decrease significantly. Although E-cadherin and desmoglein 2 proteins were stable in parental HSC-4 cells, these proteins were rapidly degraded in Snail/HSC-4 cells. The degradation of E-cadherin, but not desmoglein 2, was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis. Therefore, in HSC-4 cells Snail regulates levels of these proteins both transcriptionally and post-translationally.

IL-6 Promotes Head and Neck Tumor Metastasis by Inducing Epithelial–Mesenchymal Transition via the JAK-STAT3-SNAIL Signaling Pathway

Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions and cell polarity, resulting in the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process in head and neck cancers are poorly understood. Increasing evidence suggests that tumor microenvironment plays an important role in promoting EMT in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher Bcl-2 expression in tumor-associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a severe combined immunodeficient (SCID) mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2), when cocultured with head and neck tumor cells (CAL27), significantly enhance EMT-related changes in tumor cells predominantly by the secretion of IL-6. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or immortalized oral epithelial cells (IOE) significantly induced the expression of mesenchymal marker, vimentin, while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. These EMT-related changes were further associated with enhanced tumor and IOE cell scattering and motility. STAT3 knockdown significantly reversed IL-6-mediated tumor and IOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis in a SCID mouse xenograft model. Taken together, these results show a novel function for IL-6 in mediating EMT in head and neck tumor cells and increasing their metastatic potential.

Biological Functions of Genes in the 70-Gene MammaPrint Tumor Expression Profile Reflect Molecular Mechanism of Tumor Metastasis.

Abstract Background: MammaPrint has been validated as a reliable diagnostic tool to predict risk of distant metastasis in patients with breast cancer. In this study, we annotate the biological function of genes in the MammaPrint signature, and investigate cancer biology linking the molecular signature with respect to underlying molecular mechanism of tumor progression and tumor metastasis.Material and methods: Genes in the MammaPrint signature were translated into protein sequences. The subcellular localization, functional domain architecture and biological function of genes were annotated by using 7 different algorithms and 3 associated databases and were then interpreted within biological context. The tumor progression and tumor metastasis cascade was dissected into individual steps and the biological function of genes was subsequently associated with the tumor metastasis cascade.Results: We reveal sub cellular localization and functional domain architecture of the 70 genes in the MammaPrint signature. We find that these genes cover 12 diverse biological functional groups including metabolism; cell cycle and DNA replication; extracellular matrix adhesion and remodeling; growth, proliferation, transformation and cell death; general signal transduction and intracellular transport; growth factor; motility or actin filament organization; intracellular hydrolase; innate immune response; neuropeptide; predicted transmembrane protein with unknown function; and predicted transcriptional control or DNA binding proteins. We and demonstrate the involvement of these 70 genes in all individual steps of tumor progression and tumor metastatic cascade at both primary tumor site and metastasis site. In addition, we show that within these 70 genes, 15 genes are well known to be involved in the early embryonic development, 2 genes are known as development related transcription factors, and 3 genes (TGFB3, FGF18, WISP1) represent well characterized important epithelial-mesenchymal transition mediating TGF-β, FGF and Wnt family proteins. This is consistent with a biological model wherein tumor cells at the primary tumor site acquire metastatic ability through similar behaviors of epithelial-mesenchymal transition by turning on/off some of the same transcription factors that are used in the early embryonic development.Discussion: Our results show that genes in the MammaPrint profile cover all tumor progression and metastasis related biological processes. This finding establishes a comprehensive link between molecular signature and the underlying molecular mechanisms of tumor metastasis and explains the mechanism by which the 70-gene MammaPrint profile is predictive for the risk of distant metastasis. Further, our results support the hypothesis that the epithelial-mesenchymal transition phenomenon is a key step to conferring tumor cells with metastatic ability. Understanding underlying molecular mechanism of cancer biology reflected by the MammaPrint signature greatly improves the opportunity of applying this gene signature in clinic. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6146.

JNK2 enhances mammary carcinoma cell migration by regulating EPS8 expression and localization.

Abstract Abstract #2050 Cell migration is a tightly regulated event critical for normal development and adult wound healing, but it is also a key step in the tumor metastasis cascade. Growth factors initiate intra-cellular signaling pathways that stimulate cell movement by virtue of remodeling the actin cytoskeleton. Fine regulation of this process renders a degree of polarity to the cells, altering their cytoskeleton and providing the requisite mechanical torsion to move forward. Breast cancer cells extravasate from their primary site and travel to other organs forming metastatic colonies.Various signaling molecules including members of the Mitogen Activated Protein Kinase (MAPK) such as c-Jun N-Terminal Kinase (JNK) have been implicated in regulating cancer cell migration. How growth factors transmit signals to form dorsal ruffles and filopodia for cell migration is not precisely understood. Additionally, JNK is encoded by three genes, resulting in 10 isoforms, hence its role in metastasis is complicated. Here, we used the Polyoma Virus Middle T Antigen (PyVMT) transgenic mouse model where wildtype jnk2 was expressed or knocked out. In tumors we found that PyVMT jnk2-/- tumors express higher Epidermal Growth Factor Receptor Substrate 8 (EPS8) mRNA and protein. EPS8 was originally identified as a substrate for Epidermal Growth Factor Receptor (EGFR), and was recently reported to have an actin capping function. Hence, EPS8 is a prime candidate for connecting the EGFR signaling to actin cytoskeletion remodeling, and therefore mediate cell migration. In vitro using Boyden Chamber based migration assays, we found that the PyV MT jnk2+/+ cells migrated fivefold more than the PyV MT/jnk2-/- cells. Pan inhibition of JNK using a small peptide inhibitor TAT-JIP VII significantly reduced migration of the PyV MT/jnk2+/+ cells but was ineffective at further reducing PyVMT jnk2-/- cell migration, suggesting that the JNK2 is the major isoform to convey this function. Furthermore, shRNA mediated knock down of EPS8 in the PYVMT jnk2-/- cells, increased migration of these cells to levels comparable with the wild type counterparts. We subsequently undertook immunocytochemistry studies to evaluate EPS8 localization and cytoskeletal changes upon serum stimulation. Cells expressing JNK2 showed more dorsal ruffles upon serum stimulation, and EPS8 localized to the plasma membrane and in dorsal ruffles. In contrast, EPS8 was predominantly localized in focal adhesions in the PyVMT jnk2-/- cells, which may explain their reduced migration potential. Together our model supports an integral role for JNK2 regulation of EPS8 expression and localization to facilitate cell migration. Our current studies are aimed at further deciphering the JNK/EPS8 pathway to understand the exact molecular mechanism by which these proteins regulate a critical step in mammary cancer cell metastasis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2050.