Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.

Selective covalent targeting of GPX4 using masked nitrile-oxide electrophiles.

We recently described glutathione peroxidase 4 (GPX4) as a promising target for killing therapy-resistant cancer cells via ferroptosis. The onset of therapy resistance by multiple types of treatment results in a stable cell state marked by high levels of polyunsaturated lipids and an acquired dependency on GPX4. Unfortunately, all existing inhibitors of GPX4 act covalently via a reactive alkyl chloride moiety that confers poor selectivity and pharmacokinetic properties. Here, we report our discovery that masked nitrile-oxide electrophiles, which have not been explored previously as covalent cellular probes, undergo remarkable chemical transformations in cells and provide an effective strategy for selective targeting of GPX4. The new GPX4-inhibiting compounds we describe exhibit unexpected proteome-wide selectivity and, in some instances, vastly improved physiochemical and pharmacokinetic properties compared to existing chloroacetamide-based GPX4 inhibitors. These features make them superior tool compounds for biological interrogation of ferroptosis and constitute starting points for development of improved inhibitors of GPX4.

TRPC6 channels modulate the response of pancreatic stellate cells to hypoxia.

Pancreatic cancer is characterized by a massive fibrosis (desmoplasia), which is primarily caused by activated pancreatic stellate cells (PSCs). This leads to a hypoxic tumor microenvironment further reinforcing the activation of PSCs by stimulating their secretion of growth factors and chemokines. Since many of them elicit their effects via G-protein-coupled receptors (GPCRs), we tested whether TRPC6 channels, effector proteins of many G-protein-coupled receptor pathways, are required for the hypoxic activation of PSCs. Thus far, the function of ion channels in PSCs is virtually unexplored. qPCR revealed TRPC6 channels to be one of the most abundant TRPC channels in primary cultures of murine PSCs. TRPC6 channel function was assessed by comparing PSCs from TRPC6-/- mice and wildtype (wt) littermates. Cell migration, Ca2+ signaling, and cytokine secretion were analyzed as readout for PSC activation. Hypoxia was induced by incubating PSCs for 24 h in 1% O2 or chemically with dimethyloxalylglycine (DMOG). PSCs migrate faster in response to hypoxia. Due to reduced autocrine stimulation, TRPC6-/- PSCs fail to increase their rate of migration to the same level as wt PSCs under hypoxic conditions. This defect could not be overcome by the stimulation with platelet-derived growth factor. In line with these results, calcium influx is increased in wt but not TRPC6-/- PSCs under hypoxia. We conclude that TRPC6 channels of PSCs are major effector proteins in an autocrine stimulation pathway triggered by hypoxia.

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

Targeting fatty acid oxidation via Acyl-CoA binding protein hinders glioblastoma invasion.

The diffuse nature of Glioblastoma (GBM) tumors poses a challenge to current therapeutic options. We have previously shown that Acyl-CoA Binding Protein (ACBP, also known as DBI) regulates lipid metabolism in GBM cells, favoring fatty acid oxidation (FAO). Here we show that ACBP downregulation results in wide transcriptional changes affecting invasion-related genes. In vivo experiments using patient-derived xenografts combined with in vitro models demonstrated that ACBP sustains GBM invasion via binding to fatty acyl-CoAs. Blocking FAO mimics ACBPKD-induced immobility, a cellular phenotype that can be rescued by increasing FAO rates. Further investigation into ACBP-downstream pathways served to identify Integrin beta-1, a gene downregulated upon inhibition of either ACBP expression or FAO rates, as a mediator for ACBP's role in GBM invasion. Altogether, our findings highlight a role for FAO in GBM invasion and reveal ACBP as a therapeutic vulnerability to stall FAO and subsequent cell invasion in GBM tumors.

Origin and function of tumor stroma fibroblasts.

Tumor development is critically dependent on the formation of a supporting stroma consisting of neovasculature, inflammatory cells and activated fibroblasts. Activated fibroblasts present a heterogeneous cell population not only in regard to the expression of marker molecules but also to their origin and molecular signaling properties. The plasticity of this cell type is pointed out by the multiple transdifferentiation events that lead to the generation of activated fibroblasts which can arise from resting fibroblasts, epithelial and endothelial cells as well as from mesenchymal stem cells. Cellular in vitro and in vivo experiments have changed the perspective of fibroblasts from passive "bystanders" in the tumor microenvironment to that of important drivers of tumor progression. Here, we describe the multiple origins of fibroblast recruitment to the tumor tissue as well as the function of activated fibroblasts during tumor initiation, progression, metastasis and anti-VEGF resistance. The identification of markers present in activated fibroblasts as well as a better understanding how these cells influence other tumor compartments has led to the clinical development of anti-tumor therapies.

Quantum dot-conjugated anti-GRP78 scFv inhibits cancer growth in mice.

Semiconductor quantum dots (Qdots) have recently been shown to offer significant advantages over conventional fluorescent probes to image and study biological processes. The stability and low toxicity of QDs are well suited for biological applications. Despite this, the potential of Qdots remains limited owing to the inefficiency of existing delivery methods. By conjugating Qdots with small antibody fragments targeting membrane-bound proteins, such as GRP78, we demonstrate here that the Quantum dot- Anti-GRP78 scFv (Qdot-GRP78) retains its immunospecificity and its distribution can be monitored by visualization of multi-color fluorescence imaging both in vitro and in vivo. Moreover we demonstrate here for the first time that Qdot-GRP78 scFv bioconjugates can be efficiently internalized by cancer cells, thus upregulate phophosphate-AKT-ser473 and possess biological anti-tumour activity as shown by inhibition of breast cancer growth in a xenograft model. This suggests that nanocarrier-conjugated scFvs can be used as a therapeutic antibody for cancer treatment.

Identification of the Highly Active, Species Cross-Reactive Complex I Inhibitor BAY-179.

Mitochondria are key regulators of energy supply and cell death. Generation of ATP within mitochondria occurs through oxidative phosphorylation (OXPHOS), a process which utilizes the four complexes (complex I-IV) of the electron transport chain and ATP synthase. Certain oncogenic mutations (e.g., LKB1 or mIDH) can further enhance the reliance of cancer cells on OXPHOS for their energetic requirements, rendering cells sensitive to complex I inhibition and highlighting the potential value of complex I as a therapeutic target. Herein, we describe the discovery of a potent, selective, and species cross-reactive complex I inhibitor. A high-throughput screen of the Bayer compound library followed by hit triaging and initial hit-to-lead activities led to a lead structure which was further optimized in a comprehensive lead optimization campaign. Focusing on balancing potency and metabolic stability, this program resulted in the identification of BAY-179, an excellent in vivo suitable tool with which to probe the biological relevance of complex I inhibition in cancer indications.

Spheroid-based engineering of a human vasculature in mice.

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

Angiopoietin-2 levels are associated with disease progression in metastatic malignant melanoma.

PURPOSE: The blood vessel-destabilizing Tie2 ligand angiopoietin-2 (Ang-2) acts in concert with the vascular endothelial growth factor/vascular endothelial growth factor receptor system to control vessel assembly during tumor progression. We hypothesized that circulating soluble Ang-2 (sAng-2) may be involved in melanoma progression. EXPERIMENTAL DESIGN: Serum samples (n=98) from melanoma patients (American Joint Committee on Cancer stages I-IV), biopsies of corresponding patients, and human melanoma cell lines were analyzed for expression of Ang-2 and S100beta. Multiple sera of a subcohort of 33 patients were tested during progression from stage III to IV. Small interfering RNA-based loss-of-function experiments were done to assess effects of Ang-2 on melanoma cells. RESULTS: Circulating levels of sAng-2 correlate with tumor progression in melanoma patients (P<0.0001) and patient survival (P=0.007). Analysis of serum samples during the transition from stage III to IV identified an increase of sAng-2 up to 400%. Comparative analyses revealed a 56% superiority of sAng-2 as predictive marker over the established marker S100beta. Immunohistochemistry and reverse transcription-PCR confirmed the prominent expression of Ang-2 by tumor-associated endothelial cells but identified Ang-2 also as a secreted product of melanoma cells themselves. Corresponding cellular experiments revealed that human melanoma-isolated tumor cells were Tie2 positive and that Ang-2 acted as an autocrine regulator of melanoma cell migration and invasion. CONCLUSIONS: The experiments establish sAng-2 as a biomarker of melanoma progression and metastasis correlating with tumor load and overall survival. The identification of an autocrine angiopoietin/Tie loop controlling melanoma migration and invasion warrants further functional experiments and validate the angiopoietin/Tie system as a promising therapeutic target for human melanomas.

SLC25A32 sustains cancer cell proliferation by regulating flavin adenine nucleotide (FAD) metabolism.

SLC25A32 is a member of the solute carrier 25 family of mitochondrial transporters. SLC25A32 transports tetrahydrofolate (THF) as well as FAD into mitochondria and regulates mitochondrial one-carbon metabolism and redox balance. While it is known that cancer cells require one-carbon and FAD-dependent mitochondrial metabolism to sustain cell proliferation, the role of SLC25A32 in cancer cell growth remains unexplored. Our results indicate that the SLC25A32 gene is highly amplified in different tumors and that amplification correlates with increased mRNA expression and reduced patients´ survival. siRNA-mediated knock-down and CRISPR-mediated knock-out of SLC25A32 in cancer cells of different origins, resulted in the identification of cell lines sensitive and resistant to SLC25A32 inhibition. Mechanistically, tracing of deuterated serine revealed that SLC25A32 knock-down does not affect the mitochondrial/cytosolic folate flux as measured by Liquid Chromatography coupled Mass Spectrometry (LC-MS). Instead, SLC25A32 inhibition results in a respiratory chain dysfunction at the FAD-dependent complex II enzyme, induction of Reactive Oxygen Species (ROS) and depletion of reduced glutathione (GSH), which impairs cancer cell proliferation. Moreover, buthionine sulfoximine (BSO) treatment further sensitizes cells to ROS-mediated inhibition of cell proliferation upon SLC25A32 knock-down. Treatment of cells with the FAD precursor riboflavin and with GSH rescues cancer cell proliferation upon SLC25A32 down-regulation. Our results indicate that the reduction of mitochondrial FAD concentrations by targeting SLC25A32 has potential clinical applications as a single agent or in combination with approved cancer drugs that lead to increased oxidative stress and reduced tumor growth.

Cell surface nucleolin antagonist causes endothelial cell apoptosis and normalization of tumor vasculature.

Nucleolin is specifically transported to the surface of proliferating endothelial cells in vitro and in vivo. In contrast to its well defined functions in the nucleus and cytoplasm, the function of cell surface nucleolin is poorly defined. We have previously identified the nucleolin-binding antibody NCL3 that specifically binds to cell surface nucleolin on angiogenic blood vessels in vivo and is internalized into the cell. Here, we show that NCL3 inhibits endothelial tube formation in vitro as well as angiogenesis in the matrigel plaque assay and subcutaneous tumor models in vivo. Intriguingly, the specific targeting of proliferating endothelial cells by NCL3 in subcutaneous tumor models leads to the normalization of the tumor vasculature and as a result to an increase in tumor oxygenation. Treatment of endothelial cells with anti-nucleolin antibody NCL3 leads to a decrease of mRNA levels of the anti-apoptotic molecule Bcl-2 and as a consequence induces endothelial cell apoptosis as evidenced by PARP cleavage. These data reveal a novel mode of action for anti-angiogenic therapy and identify cell surface nucleolin as a novel target for combinatorial chemotherapy.

Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K.

Tumor development involves complex bidirectional interactions between tumor cells and host stromal cells. Endosialin (Tem1) has been identified as a highly O-glycosylated transmembrane glycoprotein, which is specifically expressed by tumor vessel-associated pericytes and stromal fibroblasts of a wide range of human tumors. Recent experiments in endosialin-deficient mice have unraveled a critical role of endosialin in site-specific tumor progression and metastasis. To molecularly understand the mechanisms of endosialin function, we aimed to identify extracellular endosialin ligands and identified Mac-2 BP/90K as a specific interaction partner. Detailed biochemical analyses identified a C-terminal fragment of Mac-2 BP/90K, which was shown to contain binding sites for galectin-3, and collagens as the structures responsible for endosialin binding. Subsequent expression analysis of Mac-2 BP/90K in vivo revealed weak or no expression in most normal tissues and strong up-regulation in tumor cells of human neoplastic tissues. Intriguingly, the expression patterns of Mac-2 BP/90K and endosialin were mutually exclusive in all human tissues. Correspondingly, loss-of-function adhesion experiments of Mac-2 BP/90K-expressing tumor cells on endosialin-expressing fibroblasts revealed a repulsive outcome of the Mac-2 BP/90K interaction. Taken together, the experiments identify a novel repulsive interaction between endosialin on stromal fibroblasts and Mac-2 BP/90K on tumor cells.

Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels.

A tumor-homing peptide, F3, selectively binds to endothelial cells in tumor blood vessels and to tumor cells. Here, we show that the cell surface molecule recognized by F3 is nucleolin. Nucleolin specifically bound to an F3 peptide affinity matrix from extracts of cultured breast carcinoma cells. Antibodies and cell surface biotin labeling revealed nucleolin at the surface of actively growing cells, and these cells bound and internalized fluorescein-conjugated F3 peptide, transporting it into the nucleus. In contrast, nucleolin was exclusively nuclear in serum-starved cells, and F3 did not bind to these cells. The binding and subsequent internalization of F3 were blocked by an antinucleolin antibody. Like the F3 peptide, intravenously injected antinucleolin antibodies selectively accumulated in tumor vessels and in angiogenic vessels of implanted "matrigel" plugs. These results show that cell surface nucleolin is a specific marker of angiogenic endothelial cells within the vasculature. It may be a useful target molecule for diagnostic tests and drug delivery applications.

The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies.

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

Lysine acetylation in mitochondria: From inventory to function.

Cellular signaling pathways are regulated in a highly dynamic fashion in order to quickly adapt to distinct environmental conditions. Acetylation of lysine residues represents a central process that orchestrates cellular metabolism and signaling. In mitochondria, acetylation seems to be the most prevalent post-translational modification, presumably linked to the compartmentation and high turnover of acetyl-CoA in this organelle. Similarly, the elevated pH and the higher concentration of metabolites in mitochondria seem to favor non-enzymatic lysine modifications, as well as other acylations. Hence, elucidating the mechanisms for metabolic control of protein acetylation is crucial for our understanding of cellular processes. Recent advances in mass spectrometry-based proteomics have considerably increased our knowledge of the regulatory scope of acetylation. Here, we review the current knowledge and functional impact of mitochondrial protein acetylation across species. We first cover the experimental approaches to identify and analyze lysine acetylation on a global scale, we then explore both commonalities and specific differences of plant and animal acetylomes and the evolutionary conservation of protein acetylation, as well as its particular impact on metabolism and diseases. Important future directions and technical challenges are discussed, and it is pointed out that the transfer of knowledge between species and diseases, both in technology and biology, is of particular importance for further advancements in this field.

Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death.

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas.

Acetyl-CoA Carboxylase 1-Dependent Protein Acetylation Controls Breast Cancer Metastasis and Recurrence.

Breast tumor recurrence and metastasis represent the main causes of cancer-related death in women, and treatments are still lacking. Here, we define the lipogenic enzyme acetyl-CoA carboxylase (ACC) 1 as a key player in breast cancer metastasis. ACC1 phosphorylation was increased in invading cells both in murine and human breast cancer, serving as a point of convergence for leptin and transforming growth factor (TGF) ß signaling. ACC1 phosphorylation was mediated by TGFß-activated kinase (TAK) 1, and ACC1 inhibition was indispensable for the elevation of cellular acetyl-CoA, the subsequent increase in Smad2 transcription factor acetylation and activation, and ultimately epithelial-mesenchymal transition and metastasis induction. ACC1 deficiency worsened tumor recurrence upon primary tumor resection in mice, and ACC1 phosphorylation levels correlated with metastatic potential in breast and lung cancer patients. Given the demonstrated effectiveness of anti-leptin receptor antibody treatment in halting ACC1-dependent tumor invasiveness, our work defines a "metabolocentric" approach in metastatic breast cancer therapy.

Identification of KCa3.1 Channel as a Novel Regulator of Oxidative Phosphorylation in a Subset of Pancreatic Carcinoma Cell Lines.

Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in KRAS as well as loss-of-function mutations in p53. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment. In the study presented here, our goal was to identify novel transporters or channels that regulate oxidative phosphorylation (OxPhos) in PDAC in order to characterize novel potential drug targets for the treatment of these cancers. We set up a Seahorse Analyzer XF based siRNA screen and identified previously described as well as novel regulators of OxPhos. The siRNA that resulted in the greatest change in cellular oxygen consumption was targeting the KCNN4 gene, which encodes for the Ca2+-sensitive K+ channel KCa3.1. This channel has not previously been reported to regulate OxPhos. Knock-down experiments as well as the use of a small molecule inhibitor confirmed its role in regulating oxygen consumption, ATP production and cellular proliferation. Furthermore, PDAC cell lines sensitive to KCa3.1 inhibition were shown to express the channel protein in the plasma membrane as well as in the mitochondria. These differences in the localization of KCa3.1 channels as well as differences in the regulation of cellular metabolism might offer opportunities for targeted therapy in subsets of PDAC.

SIAH ubiquitin ligases regulate breast cancer cell migration and invasion independent of the oxygen status.

Seven-in-absentia homolog (SIAH) proteins are evolutionary conserved RING type E3 ubiquitin ligases responsible for the degradation of key molecules regulating DNA damage response, hypoxic adaptation, apoptosis, angiogenesis, and cell proliferation. Many studies suggest a tumorigenic role for SIAH2. In breast cancer patients SIAH2 expression levels correlate with cancer aggressiveness and overall patient survival. In addition, SIAH inhibition reduced metastasis in melanoma. The role of SIAH1 in breast cancer is still ambiguous; both tumorigenic and tumor suppressive functions have been reported. Other studies categorized SIAH ligases as either pro- or antimigratory, while the significance for metastasis is largely unknown. Here, we re-evaluated the effects of SIAH1 and SIAH2 depletion in breast cancer cell lines, focusing on migration and invasion. We successfully knocked down SIAH1 and SIAH2 in several breast cancer cell lines. In luminal type MCF7 cells, this led to stabilization of the SIAH substrate Prolyl Hydroxylase Domain protein 3 (PHD3) and reduced Hypoxia-Inducible Factor 1α (HIF1α) protein levels. Both the knockdown of SIAH1 or SIAH2 led to increased apoptosis and reduced proliferation, with comparable effects. These results point to a tumor promoting role for SIAH1 in breast cancer similar to SIAH2. In addition, depletion of SIAH1 or SIAH2 also led to decreased cell migration and invasion in breast cancer cells. SIAH knockdown also controlled microtubule dynamics by markedly decreasing the protein levels of stathmin, most likely via p27(Kip1). Collectively, these results suggest that both SIAH ligases promote a migratory cancer cell phenotype and could contribute to metastasis in breast cancer.