Uncovering the signaling landscape controlling breast cancer cell migration identifies novel metastasis driver genes.

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.

IGF1R signaling drives antiestrogen resistance through PAK2/PIX activation in luminal breast cancer.

Antiestrogen resistance in estrogen receptor positive (ER+) breast cancer is associated with increased expression and activity of insulin-like growth factor 1 receptor (IGF1R). Here, a kinome siRNA screen has identified 10 regulators of IGF1R-mediated antiestrogen with clinical significance. These include the tamoxifen resistance suppressors BMPR1B, CDK10, CDK5, EIF2AK1, and MAP2K5, and the tamoxifen resistance inducers CHEK1, PAK2, RPS6KC1, TTK, and TXK. The p21-activated kinase 2, PAK2, is the strongest resistance inducer. Silencing of the tamoxifen resistance inducing genes, particularly PAK2, attenuates IGF1R-mediated resistance to tamoxifen and fulvestrant. High expression of PAK2 in ER+ metastatic breast cancer patients is correlated with unfavorable outcome after first-line tamoxifen monotherapy. Phospho-proteomics has defined PAK2 and the PAK-interacting exchange factors PIXα/ß as downstream targets of IGF1R signaling, which are independent from PI3K/ATK and MAPK/ERK pathways. PAK2 and PIXα/ß modulate IGF1R signaling-driven cell scattering. Targeting PIXα/ß entirely mimics the effect of PAK2 silencing on antiestrogen re-sensitization. These data indicate PAK2/PIX as an effector pathway in IGF1R-mediated antiestrogen resistance.

Alternative signaling network activation through different insulin receptor family members caused by pro-mitogenic antidiabetic insulin analogues in human mammary epithelial cells.

INTRODUCTION: Insulin analogues are designed to have improved pharmacokinetic parameters compared to regular human insulin. This provides a sustained control of blood glucose levels in diabetic patients. All novel insulin analogues are tested for their mitogenic side effects, however these assays do not take into account the molecular mode of action of different insulin analogues. Insulin analogues can bind the insulin receptor and the insulin-like growth factor 1 receptor with different affinities and consequently will activate different downstream signaling pathways. METHODS: Here we used a panel of MCF7 human breast cancer cell lines that selectively express either one of the isoforms of the INSR or the IGF1R. We applied a transcriptomics approach to assess the differential transcriptional programs activated in these cells by either insulin, IGF1 or X10 treatment. RESULTS: Based on the differentially expressed genes between insulin versus IGF1 and X10 treatment, we retrieved a mitogenic classifier gene set. Validation by RT-qPCR confirmed the robustness of this gene set. The translational potential of these mitogenic classifier genes was examined in primary human mammary cells and in mammary gland tissue of mice in an in vivo model. The predictive power of the classifier genes was evaluated by testing all commercial insulin analogues in the in vitro model and defined X10 and glargine as the most potent mitogenic insulin analogues. CONCLUSIONS: We propose that these mitogenic classifier genes can be used to test the mitogenic potential of novel insulin analogues as well as other alternative molecules with an anticipated affinity for the IGF1R.

Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant.

Tumor cell migration is a key process for cancer cell dissemination and metastasis that is controlled by signal-mediated cytoskeletal and cell matrix adhesion remodeling. Using a phagokinetic track assay with migratory H1299 cells, we performed an siRNA screen of almost 1,500 genes encoding kinases/phosphatases and adhesome- and migration-related proteins to identify genes that affect tumor cell migration speed and persistence. Thirty candidate genes that altered cell migration were validated in live tumor cell migration assays. Eight were associated with metastasis-free survival in breast cancer patients, with integrin ß3-binding protein (ITGB3BP), MAP3K8, NIMA-related kinase (NEK2), and SHC-transforming protein 1 (SHC1) being the most predictive. Examination of genes that modulate migration indicated that SRPK1, encoding the splicing factor kinase SRSF protein kinase 1, is relevant to breast cancer outcomes, as it was highly expressed in basal breast cancer. Furthermore, high SRPK1 expression correlated with poor breast cancer disease outcome and preferential metastasis to the lungs and brain. In 2 independent murine models of breast tumor metastasis, stable shRNA-based SRPK1 knockdown suppressed metastasis to distant organs, including lung, liver, and spleen, and inhibited focal adhesion reorganization. Our study provides comprehensive information on the molecular determinants of tumor cell migration and suggests that SRPK1 has potential as a drug target for limiting breast cancer metastasis.

Silencing of doublecortin-like (DCL) results in decreased mitochondrial activity and delayed neuroblastoma tumor growth.

Doublecortin-like (DCL) is a microtubule-binding protein crucial for neuroblastoma (NB) cell proliferation. We have investigated whether the anti-proliferative effect of DCL knockdown is linked to reduced mitochondrial activity. We found a delay in tumor development after DCL knockdown in vivo in doxycycline-inducible NB tumor xenografts. To understand the mechanisms underlying this tumor growth retardation we performed a series of in vitro experiments in NB cell lines. DCL colocalizes with mitochondria, interacts with the mitochondrial outer membrane protein OMP25/ SYNJ2BP and DCL knockdown results in decreased expression of genes involved in oxidative phosphorylation. Moreover, DCL knockdown decreases cytochrome c oxidase activity and ATP synthesis. We identified the C-terminal Serine/Proline-rich domain and the second microtubule-binding area as crucial DCL domains for the regulation of cytochrome c oxidase activity and ATP synthesis. Furthermore, DCL knockdown causes a significant reduction in the proliferation rate of NB cells under an energetic challenge induced by low glucose availability. Together with our previous studies, our results corroborate DCL as a key player in NB tumor growth in which DCL controls not only mitotic spindle formation and the stabilization of the microtubule cytoskeleton, but also regulates mitochondrial activity and energy availability, which makes DCL a promising molecular target for NB therapy.

Two-photon intravital multicolour imaging to study metastatic behaviour of cancer cells in vivo.

In the last decade, intravital microscopy on breast tumours in mice at single-cell resolution has resulted in important new insight into mechanisms of metastatic behaviour such as migration, invasion, and intravasation of tumour cells; angiogenesis; and the response of immune cells. This chapter describes the methods that can be used for analysing tumour cell motility in a mouse model of breast cancer metastasis. It includes protocols for generation of a labelled primary tumour, its imaging with two-photon microscopy, and the processing of time-lapse image data. Furthermore, we present a methodology, recently developed in our laboratory that combines multicolour imaging with an inducible cell model to study the role of a specific gene of interest in tumour cell motility in vivo. This protocol can be used to image the metastatic behaviour of different individual tumour cells within the same tumour microenvironment and correlate it with metastasis formation. Additional protocols for labelling macrophages to visualise blood flow and image analysis are also included.

Two-photon intravital multicolor imaging combined with inducible gene expression to distinguish metastatic behavior of breast cancer cells in vivo.

PURPOSE: The aim of this study is to use multicolor intravital imaging together with an inducible cell model to compare metastatic behavior of control and genetically modified breast cancer cell populations within the intact primary tumor of a mouse. PROCEDURE: GFP-MTLn3-ErbB1 cells were generated with doxycycline-regulated conditional transgene expression using lentiviral TREAutoR3-cyan fluorescent protein (CFP). CFP expression together with tumor cell motility is monitored in vitro and in vivo. RESULTS: Effective and tight control of doxycycline-induced CFP expression was observed both in vitro and in vivo. Intravital multiphoton microscopy on intact orthotopic tumors allowed a clear discrimination between GFP-only and (GFP + CFP) cell populations, which enables direct comparison of the motility behavior of two different cell populations in the same microenvironment in vivo. CONCLUSIONS: This system is robust and versatile for conditional gene expression and can be used to study the role of individual candidate metastasis genes in vitro and in vivo. This technology will allow investigations of cellular events in cancer metastasis and in particular intravasation within a primary tumor.

Annexin A1 regulates TGF-beta signaling and promotes metastasis formation of basal-like breast cancer cells.

Annexin A1 (AnxA1) is a candidate regulator of the epithelial- to mesenchymal (EMT)-like phenotypic switch, a pivotal event in breast cancer progression. We show here that AnxA1 expression is associated with a highly invasive basal-like breast cancer subtype both in a panel of human breast cancer cell lines as in breast cancer patients and that AnxA1 is functionally related to breast cancer progression. AnxA1 knockdown in invasive basal-like breast cancer cells reduced the number of spontaneous lung metastasis, whereas additional expression of AnxA1 enhanced metastatic spread. AnxA1 promotes metastasis formation by enhancing TGFbeta/Smad signaling and actin reorganization, which facilitates an EMT-like switch, thereby allowing efficient cell migration and invasion of metastatic breast cancer cells.

An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2(-/-) gammac (-/-) mouse.

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2(-/-)gammac(-/-) mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2(-/-) gammac(-/-) mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation.

Extracellular signal-regulated kinase activation during renal ischemia/reperfusion mediates focal adhesion dissolution and renal injury.

Acute renal failure due to ischemia/reperfusion involves disruption of integrin-mediated cellular adhesion and activation of the extracellular signal-regulated kinase (ERK) pathway. The dynamics of focal adhesion organization and phosphorylation during ischemia/reperfusion in relation to ERK activation are unknown. In control kidneys, protein tyrosine-rich focal adhesions, containing focal adhesion kinase, paxillin, and talin, were present at the basolateral membrane of tubular cells and colocalized with short F-actin stress fibers. Unilateral renal ischemia/reperfusion caused a reversible protein dephosphorylation and loss of focal adhesions. The focal adhesion protein phosphorylation rebounded in a biphasic manner, in association with increased focal adhesion kinase, Src, and paxillin tyrosine phosphorylation. Preceding phosphorylation of these focal adhesion proteins, reperfusion caused increased phosphorylation of ERK. The specific mitogen-activated protein kinase kinase 1/2 inhibitor U0126 prevented ERK activation and attenuated focal adhesion kinase, paxillin, and Src phosphorylation, focal adhesion restructuring, and ischemia/reperfusion-induced renal injury. We propose a model whereby ERK activation enhanced protein tyrosine phosphorylation during ischemia/reperfusion, thereby driving the dynamic dissolution and restructuring of focal adhesions and F-actin cytoskeleton during reperfusion and renal injury.

Peptide-bond modified glutathione conjugate analogs modulate GSTpi function in GSH-conjugation, drug sensitivity and JNK signaling.

Glutathione S-transferase pi (GST, E.C.2.5.1.18) overexpression contributes to resistance of cancer cells towards cytostatic drugs. Furthermore, GSTpi is involved in the cellular stress response through inhibition of Jun N-terminal-kinase (JNK), a process that can be modulated by GST inhibitors. GSH conjugates are potent GST inhibitors, but are sensitive towards gamma-glutamyltranspeptidase (gammaGT)-mediated breakdown. In search for new peptidase stable GST inhibitors we employed the following strategy: (1) selection of a suitable (GST inhibiting) peptide-bond isostere from a series of previously synthesized gammaGT stabilized GSH-analogs. (2) The use of this peptidomimetic strategy to prepare a GSTpi selective inhibitor. Two gammaGT stable GSH conjugate analogs inhibited human GSTs, although non-selectively. One of these, a urethane-type peptide-bond is well accepted by GSTs and we selected this modification for the development of a gammaGT stable, GSTpi selective inhibitor, UrPhg-Et(2). This compound displayed selectivity for GSTpi compared to alpha and mu class enzymes. Furthermore, the inhibitor reversed GSTpi-mediated drug resistance (MDR) in breast tumor cells. In addition, short-term exposure of cells to UrPhg-Et(2) led to GSTpi oligomerization and JNK activation, suggesting that it activates the JNK-cJun signaling module through GSTpi dissociation. Altogether, we show the successful use of peptidomimetic glutathione conjugate analogs as GST inhibitors and MDR-modifiers. As many MDR related enzymes, such as MRP1, glyoxalase 1 and DNA-pk are also inhibited by GSH conjugates, these peptidomimetic compounds can be used as scaffolds for the development of multi-target MDR drugs.