Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelialto-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imagingbased experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.carcinoma | receptor tyrosine kinase | breast cancer

Epithelial-to-mesenchymal transition (EMT) is a critical event in the progression toward cancer metastasis. The intermediate filament protein vimentin is an important marker of EMT and a requisite regulator of mesenchymal cell migration. However, it is not known how vimentin functionally contributes to cancer cell invasion. Here, we report that ectopic expression of oncogenic H-Ras-V12G and Slug induces vimentin expression and migration in pre-malignant breast epithelial cells. Conversely, vimentin expression is necessary for Slug-or H-Ras-V12G-induced EMT-associated migration. Furthermore, silencing of vimentin in breast epithelial cells results in specific changes in invasiveness-related gene expression including upregulation of RAB25 (small GTPase Rab25) and downregulation of AXL (receptor tyrosine kinase Axl), PLAU (plasminogen activator, urokinase) and ITGB4 (integrin b4-subunit). Importantly, gene expression profiling analyses reveal that vimentin expression correlates positively/ negatively with these genes also in multiple breast cancer cell lines and breast cancer patient samples. Focusing on the tyrosine kinase Axl, we show that induction of vimentin by EMT is associated with upregulation of Axl expression and that Axl enhances the migratory activity of pre-malignant breast epithelial cells. Using null and knock-down cells and overexpression models, we also show that regulation of breast cancer cell migration in two-and three-dimensional matrices by vimentin is Axldependent and that Axl functionally contributes to lung extravasation of breast cancer cells in mice. In conclusion, our data show that vimentin functionally contributes to EMT and is required for induction of Axl expression. Moreover, these results provide a molecular explanation for vimentin-dependent cancer cell migration during EMT by identifying Axl as a key proximal component in this process.

Despite the differential expression of S100A14 (a newly identified S100 member) in various human cancers including oral squamous cell carcinomas (OSCCs), its biological role in tumour invasion has not been characterised. The aim of this study was thus to investigate the possible role of S100A14 in OSCC cell invasion. Using immunohistochemistry in normal (n=13), dysplastic (n=10) and OSCC (n=16) archival tissues, S100A14 protein was found to be down-regulated/lost with concomitant membrane to cytoplasmic translocation in OSCCs, especially in the invading tumour islands. These expression data were corroborated by profiling S100A14 mRNA expression using quantitative RT-PCR (qRT-PCR) in an in vitro human OSCC progression model consisting of cell-lines derived from normal (n=3), dysplastic (n=3) and OSCC (n=8) tissues. Employing in vitro Matrigel invasion assay, we demonstrated that retroviral vector mediated over-expression of S100A14 resulted in significant decrease in the invasive potential of OSCC derived CaLH3 and H357 cell-lines whereas siRNA mediated knockdown resulted in significant increase in the invasive potential of CaLH3 cell-line. Pathway focused PCR array and validation using qRT-PCR revealed that S100A14 over-expression was associated with down-regulation of MMP1 and MMP9 mRNAs in both CaLH3 and H357 cell-lines. Further, S100A14 over-expression was found to be associated with suppression of MMP9 gelatinolytic activity in CaLH3 cell-line. Additionally, an inverse correlation between mRNA expression levels of MMP1 and MMP9 with S100A14 was found in 19 cases of OSCCs. Collectively, these data provide the first evidence for a role of S100A14 protein in regulation of OSCC cell invasion by modulating expression of MMP1 and MMP9.

The transcription factor p63 is central for epithelial homeostasis and development. In our model of epithelial to mesenchymal transition (EMT) in human prostate cells, p63 was one of the most down-regulated transcription factors during EMT. We therefore investigated the role of p63 in EMT. Over-expression of the predominant epithelial isoform ΔNp63α in mesenchymal type cells of the model led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). This was corroborated by a reciprocal effect when p63 was knocked down in epithelial EP156T cells. Global gene expression analyses showed that ΔNp63α induced gene modules involved in both cell-to-cell and cell-to-extracellular-matrix junctions in mesenchymal type cells. Genome-wide analysis of p63 binding sites using ChIP-seq analyses confirmed binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells. DH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knock-down of these factors, respectively, were not sufficient alone or in combination with ΔNp63α to reverse completely the mesenchymal phenotype. The partial reversion of epithelial to mesenchymal transition might reflect the ability of ΔNp63α, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The utility of ΔNp63α expression and the potential of reduced EMP in order to counteract metastasis warrant further investigation.

Axl is a member of the TAM (Tyro3, Axl and Mer) family of receptor tyrosine kinases that regulate multiple cellular responses including cell survival, proliferation, and migration. Axl expression is predictive of poor patient overall survival in a variety of human cancers including triple negative breast (TNBC), pancreatic ductal adenocarcinoma (PDA) and non-small cell lung cancer (NSCLC). Axl expression is induced by the epithelial-to-mesenchymal transition (EMT) gene program in cancer cells and Axl signaling is required to maintain EMT-associated features including invasiveness, metastasis, stem cell-like traits and resistance to targeted inhibitors and other chemotherapeutic agents. BGB324/R428 is an oral, selective small molecule inhibitor of Axl that recently was evaluated in early clinical safety studies in healthy volunteers. Treatment with BGB324 up to and including 1.5 gms daily (per os) was established as being safe and well tolerated. The endpoints of the study included pharmacokinetics and safety. Bioavailability was increased in the presence of food and systemic exposure increased dose proportionately. At the highest dose the apparent elimination half-life approached four days presenting a range of dosing options. We evaluated the effects of BGB324 in preclinical models of TNBC, PDA and NSCLC, including 2D/3D cell culture and mouse xenograft models, in combination with targeted and chemotherapeutic agents. BGB324 treatment of mesenchymal carcinoma cells blocked invasiveness and enhanced chemotherapeutic efficacy. BGB324 abrogated the tumor initiation capacity of TNBC cells, an activity associated with cancer stem cells. BGB324 treatment blocked the emergence of EMT-associated acquired resistance to erlotinib in human NSCLC xenografts. Furthermore, combination treatment of BGB324 with chemotherapy inhibited the growth of human NSCLC xenografts and significantly prolonged survival in orthotopic and genetically engineered mouse models of PDA. Collectively, these data suggest that the first-in-class selective Axl inhibitor BGB324 can overcome EMT-related acquired therapeutic resistance and enhance the efficacy of multiple anti-cancer strategies. Together with the results of results of the early clinical safety studies, this provides a rationale for further clinical studies. Citation Format: Katarzyna Wnuk-Lipinska, Crina Tiron, Gro Gausdal, Tone Sandal, Robin Frink, Stefan Hinz, Monica Hellesøy, Lavina Ahmed, Hallvard Haugen, Xiao Liang, Magnus Blø, David Micklem, Murray Yule, John Minna, Longen Zhou, Rolf Brekken, James Lorens. BGB324, a selective small molecule Axl kinase inhibitor to overcome EMT-associated drug resistance in carcinomas: Therapeutic rationale and early clinical studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1747. doi:10.1158/1538-7445.AM2014-1747

Metastasis underlies the majority of cancer-related deaths. Hence, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMT) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in mammography-detected primary breast cancers independently predicts strongly reduced overall patient survival, and matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by epithelial-to-mesenchymal transition in pre-malignant mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Using epi-allelic RNA interference analysis in metastatic breast cancer cells we delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness, and to form tumors in foreign and tissue engineered microenvironments in vivo. Importantly, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs, and increased overall survival, in two different optical imaging-based experimental breast cancer models. Thus, Axl represents a novel downstream effector of tumor cell EMT that is required for breast cancer metastasis. The detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B105.

A patient with multiple myeloma is reported with severe hypogammaglobulinaemia but without monoclonal immunoglobulin (M-component) in the serum or Bence Jones protein in the urine. In May-Griinwald-Giernsa stained bone marrow film 45 % of the cells resembled plasma cells. Marrow immunofluorescent studies showed no increase of immunoglobulin containing cells. Ultrastructural studies showed a large number of cells with a well developed rough endoplasmic reticulum and other morphological features characteristic for plasma cells. Stavem P (1967) A new bone marrow biopsy Whicher J T, Davies J D & Grayburn J A (1975) needle. Scand J Haemarol 4, 158-60.Intact and fragmented intracellular immunoglobulin in a case of non-secretory myeloma. J Clin Pathol 28, 5459.

Axl is a member of the TAM (Tyro3, Axl and Mer) family of receptor tyrosine kinases that regulate multiple cellular responses including cell survival, proliferation, and migration. Axl expression is associated with a variety of human cancers including non-small cell lung carcinoma (NSCLC), and is predictive of poor patient overall survival. Axl is induced by the epithelial-to-mesenchymal transition (EMT) gene program in cancer cells. Axl signaling is required to maintain EMT-associated features including invasiveness, metastasis, and can confer resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), as well as other chemotherapeutic agents. BGB324 is a potent, reversible and selective small molecule inhibitor of Axl that has recently entered into phase I clinical trials. We evaluated the effects of BGB324 on NSCLC cells in in vitro 3D assays and in mouse xenograft models, in combination with targeted and chemotherapeutic agents. BGB324 in combination with EGFR TKI erlotinib demonstrated a synergistic anti-proliferation effect on NCI-H1299 (mesenchymal, EGFR wild-type, erlotinib-resistant) human NSCLC cells in 3D culture. In mouse xenograft models using NCI-H1299 cells BGB324 treatment significantly enhanced the antitumor acitivity of Docetaxel. Similarly, using the mesenchymal NSCLC cell line A549 (EGFR wild-type), the combination of BGB324 with either erlotinib or anti-VEGF agent bevacizumab showed synergistic or additive anti-tumour activity. Furthermore, in HCC827 (EGFR mutant, erlotinib-sensitive) human NSCLC xenograft model, addition of BGB324 treatment strikely delayed the emergence of acquired resistance to erlotinib. Taken together, these data suggest the first-in-class selective Axl inhibitor BGB324 can overcome acquired resistance in in vivo models of NSCLC. Studies in human NSCLC patient-derived xenograft models and mechanisms of action are in progress. Citation Format: Katarzyna Wnuk-Lipinska, Gro Gausdal, Tone Sandal, Robin Frink, Stefan Hinz, Monica Hellesøy, Lavina Ahmed, Hallvard Haugen, Hallvard Haugen, Liang Xiao, Magnus Blø, David Micklem, John Minna, Rolf Brekken, Longen Zhou, James Lorens. Selective small molecule AXL inhibitor BGB324 overcomes acquired drug resistance in non-small cell lung carcinoma models. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B30.