ABSTRACT
Over the past 20â years, the Ste20-like kinase (SLK; also known as STK2) has emerged as a central regulator of cytoskeletal dynamics. Reorganization of the cytoskeleton is necessary for a plethora of biological processes including apoptosis, proliferation, migration, tissue repair and signaling. Several studies have also uncovered a role for SLK in disease progression and cancer. Here, we review the recent findings in the SLK field and summarize the various roles of SLK in different animal models and discuss the biochemical mechanisms regulating SLK activity. Together, these studies have revealed multiple roles for SLK in coupling cytoskeletal dynamics to cell growth, in muscle repair and in negative-feedback loops critical for cancer progression. Furthermore, the ability of SLK to regulate some systems appears to be kinase activity independent, suggesting that it may be an important scaffold for signal transduction pathways. These various findings reveal highly complex functions and regulation patterns of SLK in development and disease, making it a potential therapeutic target.
Subject(s)
Apoptosis , Signal Transduction , Animals , Cytoskeleton , Microtubules , PhosphorylationABSTRACT
BACKGROUND: Approximately 5-10% of HER2-positive breast cancers can be defined by low expression of the Ste20-like kinase, SLK, and high expression of SOX10. Our lab has observed that genetic deletion of SLK results in the induction of Sox10 and significantly accelerates tumor initiation in a HER2-induced mammary tumor model. However, the mechanism responsible for the induction of SOX10 gene expression in this context remains unknown. METHODS: Using tumor-derived cell lines from MMTV-Neu mice lacking SLK and biochemical approaches, we have characterized the signaling mechanisms and relevant DNA elements driving Sox10 expression. RESULTS: Biochemical and genetic analyses of the SOX10 regulatory region in SLK-deficient mammary tumor cells show that Sox10 expression is dependent on a novel -7kb enhancer that harbors three SoxE binding sites. ChIP analyses demonstrate that Sox9 is bound to those elements in vivo. Our data show that AKT can directly phosphorylate Sox9 in vitro at serine 181 and that AKT inhibition blocks Sox9 phosphorylation and Sox10 expression in SLK(-/-) tumor cells. AKT-mediated Sox9 phosphorylation increases its transcriptional activity on the Sox10 -7kb enhancer without altering its DNA-binding activity. Interestingly, analysis of murine and human mammary tumors reveals a direct correlation between the levels of active phospho-Sox9 S181 and Sox10 expression. CONCLUSIONS: Our results have identified a novel Sox10 enhancer and validated Sox9 as a direct target for AKT. As Sox10 is a biomarker for triple-negative breast cancers (TNBC), these findings might have major implications in the targeting and treatment of those cancers.
Subject(s)
Breast Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, ErbB-2/metabolism , SOX9 Transcription Factor/metabolism , SOXE Transcription Factors/genetics , Animals , Binding Sites , Breast Neoplasms/genetics , Cell Line, Tumor , Disease Models, Animal , Enhancer Elements, Genetic , Female , Humans , Mice , Mice, Transgenic , Phosphorylation , Protein Serine-Threonine Kinases/deficiency , Transcription, GeneticABSTRACT
BACKGROUND: Breast cancer is a highly heterogeneous disease with multiple drivers and complex regulatory networks. Periostin (Postn) is a matricellular protein involved in a plethora of cancer types and other diseases. Postn has been shown to be involved in various processes of tumor development, such as angiogenesis, invasion, cell survival and metastasis. The expression of Postn in breast cancer cells has been correlated with a more aggressive phenotype. Despite extensive research, it remains unclear how epithelial cancer cells regulate Postn expression. METHODS: Using murine tumor models and human TMAs, we have assessed the proportion of tumor samples that have acquired Postn expression in tumor cells. Using biochemical approaches and tumor cell lines derived from Neu+ murine primary tumors, we have identified major regulators of Postn gene expression in breast cancer cell lines. RESULTS: Here, we show that, while the stromal compartment typically always expresses Postn, about 50% of breast tumors acquire Postn expression in the epithelial tumor cells. Furthermore, using an in vitro model, we show a cross-regulation between FGFR, TGFß and PI3K/AKT pathways to regulate Postn expression. In HER2-positive murine breast cancer cells, we found that basic FGF can repress Postn expression through a PKC-dependent pathway, while TGFß can induce Postn expression in a SMAD-independent manner. Postn induction following the removal of the FGF-suppressive signal is dependent on PI3K/AKT signaling. CONCLUSION: Overall, these results reveal a novel regulatory mechanism and shed light on how breast tumor cells acquire Postn expression. This complex regulation is likely to be cell type and cancer specific as well as have important therapeutic implications.
Subject(s)
Breast Neoplasms/genetics , Cell Adhesion Molecules/genetics , Gene Expression Regulation, Neoplastic , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Transforming Growth Factor beta/metabolism , Animals , Breast Neoplasms/metabolism , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Epithelial Cells/metabolism , Female , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Receptor, ErbB-2/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacologyABSTRACT
The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover and is regulated by the LIM domain-binding proteins Ldb1 and Ldb2. These adapter proteins have been demonstrated to interact with LMO4 in the organization of transcriptional complexes. Therefore, we have assessed the ability of LMO4 to also interact and regulate SLK activity. Our data show that LMO4 can directly bind to SLK and activate its kinase activity in vitro and in vivo. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding and Cre-mediated deletion of LMO4 in conditional LMO4(fl/fl) fibroblasts inhibits cell migration and SLK activation. Deletion of LMO4 impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Supporting this, Src/Yes/Fyn-deficient cells (SYF) expressing very low levels of LMO4 do not recruit SLK to the leading edge. Re-expression of wildtype Myc-LMO4 in SYF cells, but not a mutant version, restores SLK localization and kinase activity. Overall, our data suggest that activation of SLK by haptotactic signals requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. Furthermore, this establishes a novel cytosolic role for the transcriptional co-activator LMO4.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Movement , Fibroblasts/cytology , Fibroblasts/enzymology , LIM Domain Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Pseudopodia/metabolism , src-Family Kinases/metabolism , 3T3 Cells , Adaptor Proteins, Signal Transducing/chemistry , Animals , DNA-Binding Proteins/metabolism , Enzyme Activation , Female , Gene Deletion , HEK293 Cells , Humans , LIM Domain Proteins/chemistry , Mice , Protein Binding , Protein Structure, Tertiary , Protein Transport , Subcellular Fractions/metabolismABSTRACT
INTRODUCTION: Periostin (Postn) is a secreted cell adhesion protein that activates signaling pathways to promote cancer cell survival, angiogenesis, invasion, and metastasis. Interestingly, Postn is frequently overexpressed in numerous human cancers, including breast, lung, colon, pancreatic, and ovarian cancer. METHODS: Using transgenic mice expressing the Neu oncogene in the mammary epithelium crossed into Postn-deficient animals, we have assessed the effect of Postn gene deletion on Neu-driven mammary tumorigenesis. RESULTS: Although Postn is exclusively expressed in the stromal fibroblasts of the mammary gland, Postn deletion does not affect mammary gland outgrowth during development or pregnancy. Furthermore, we find that loss of Postn in the mammary epithelium does not alter breast tumor initiation or growth in mouse mammary tumor virus (MMTV)-Neu expressing mice but results in an apocrine-like tumor phenotype. Surprisingly, we find that tumors derived from Postn-null animals express low levels of Notch protein and Hey1 mRNA but increased expression of androgen receptor (AR) and AR target genes. We show that tumor cells derived from wild-type animals do not proliferate when transplanted in a Postn-null environment but that this growth defect is rescued by the overexpression of active Notch or the AR target gene prolactin-induced protein (PIP/GCDFP-15). CONCLUSIONS: Together our data suggest that loss of Postn in an ErbB2/Neu/HER2 overexpression model results in apocrine-like tumors that activate an AR-dependent pathway. This may have important implications for the treatment of breast cancers involving the therapeutic targeting of periostin or Notch signaling.
Subject(s)
Cell Adhesion Molecules/deficiency , Receptor, ErbB-2/genetics , Receptor, ErbB-2/metabolism , Receptor, Notch1/metabolism , Receptors, Androgen/metabolism , Sweat Gland Neoplasms/genetics , Sweat Gland Neoplasms/metabolism , Animals , Apocrine Glands/metabolism , Apocrine Glands/pathology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Disease Models, Animal , Female , Gene Expression , Genotype , Humans , Immunohistochemistry , Mammary Glands, Animal/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Phenotype , Sweat Gland Neoplasms/mortality , Sweat Gland Neoplasms/pathology , Tumor BurdenABSTRACT
BACKGROUND: Over the past decade, the Ste20-like kinase SLK, has been implicated in several signaling processes. SLK repression has been shown to impair cell cycle kinetics and inhibit FAK-mediated cell migration. Here, using a gene trapped allele, we have generated mice expressing a truncated form of the SLK kinase. RESULTS: Our results show that an SLK-LacZ fusion protein is expressed in embryonic stem cells and in embryos throughout development. We find that the SLK-LacZ fusion protein is less efficient at phosphorylating substrates resulting in reduced cell proliferation within the embryos and angiogenic defects in the placentae of the homozygous mutant animals at embryonic day (E) 12.5. This results in marked developmental defects and apoptotic lesions in the embryos by E14.5. CONCLUSIONS: Homozygotes expressing the SLK-LacZ fusion protein present with an embryonic lethal phenotype occurring between E12.5 and E14.5. Overall, we demonstrate a requirement for SLK kinase activity in the developing embryo and placenta.
Subject(s)
Embryo, Mammalian/enzymology , Embryonic Development/physiology , Placenta/enzymology , Pregnancy Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Embryo, Mammalian/cytology , Female , Mice , Mice, Transgenic , Placenta/cytology , Pregnancy , Pregnancy Proteins/genetics , Protein Serine-Threonine Kinases/geneticsABSTRACT
SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte-specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins-paxillin, vinculin, and talin-1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin-1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin-induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin-1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin-1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.
Subject(s)
Nephrosis , Podocytes , Mice , Animals , Podocytes/metabolism , Paxillin/metabolism , Vinculin/metabolism , Talin/genetics , Talin/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Doxorubicin/toxicity , Protein Serine-Threonine Kinases/metabolismABSTRACT
Kinases are known to have kinase activity independent functions. To gain further insights into potential kinase-independent functions of SLK/STK2, we have developed a kinase-dead allele, SLKK63R using in vivo CRISPR/Cas technology. Our studies show that blastocysts homozygote for SLKK63R do not develop into viable mice. However, heterozygotes are viable and fertile with no overt phenotypes. Analyses of mouse embryonic fibroblasts show that expression of SLKK63R results in a 50% decrease in kinase activity in heterozygotes. In contrast to previous studies, our data show that SLK does not form homodimers and that the kinase defective allele does not act in a dominant negative fashion. Expression of SLKK63R leads to altered Rac1 and RhoA activity, increased stress fiber formation and delayed focal adhesion turnover. Our data support a previously observed role for SLK in cell migration and suggest that at least 50% kinase activity is sufficient for embryonic development.
ABSTRACT
While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.
Subject(s)
B7-H1 Antigen , Interferon Type I , Oncolytic Virotherapy , Oncolytic Viruses , Animals , Female , Humans , Mice , B7-H1 Antigen/metabolism , B7-H1 Antigen/immunology , B7-H1 Antigen/genetics , Cell Line, Tumor , Glycolysis , Interferon Type I/metabolism , Interferon Type I/immunology , Lactic Acid/metabolism , Neoplasms/immunology , Neoplasms/therapy , Neoplasms/metabolism , Oncolytic Virotherapy/methods , Oncolytic Viruses/physiology , Signal Transduction , MaleABSTRACT
Null mutations in the pea3 allele compromise the capacity of mammary tumors to metastasize in MMTV-Neu/ErbB2/HER2 transgenic mice, indicating a motility defect in PEA3-null cells. Cellular and biochemical analyses of established PEA3-null fibroblasts show impaired motility and aberrant localization of adhesion proteins in spreading cells. Our results show that PEA3-/- cells express normal levels of key adhesion components, but that spreading PEA3-null cells fail to activate c-src and to downregulate phospho-FAK(Y397), suggesting that focal adhesion signaling is impaired. Supporting this, biochemical analysis revealed that adhesion complex-associated proteins such as p130Cas failed to undergo tyrosine phosphorylation and dissociated from the adhesion complex with delayed kinetics. Overall our data show that the motility defects observed in PEA3-null cells are due to altered adhesion signaling.
Subject(s)
Cell Movement/physiology , Crk-Associated Substrate Protein/metabolism , Fibroblasts/metabolism , Genes, src/physiology , Transcription Factors/physiology , Animals , Blotting, Western , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , Fibroblasts/cytology , Fibronectins/pharmacology , Fluorescent Antibody Technique , Focal Adhesions/physiology , Immunoprecipitation , Mammary Tumor Virus, Mouse/genetics , Mice , Mice, Knockout , Mice, Transgenic , Phosphorylation , Receptor, ErbB-2/genetics , Signal Transduction , Tyrosine/metabolismABSTRACT
Targeted therapy resistance frequently develops in melanoma due to intratumor heterogeneity and epigenetic reprogramming. This also typically induces cross-resistance to immunotherapies. Whether this includes additional modes of therapy has not been fully assessed. We show that co-treatments of MAPKi with VSV-based oncolytics do not function in a synergistic fashion; rather, the MAPKis block infection. Melanoma resistance to vemurafenib further perturbs the cells' ability to be infected by oncolytic viruses. Resistance to vemurafenib can be induced by the loss of SOX10, a common proliferative marker in melanoma. The loss of SOX10 promotes a cross-resistant state by further inhibiting viral infection and replication. Analysis of RNA-seq datasets revealed an upregulation of interferon-stimulated genes (ISGs) in SOX10 knockout populations and targeted therapy-resistant cells. Interestingly, the induction of ISGs appears to be independent of type I IFN production. Overall, our data suggest that the pathway mediating oncolytic resistance is due to the loss of SOX10 during acquired drug resistance in melanoma.
Subject(s)
Melanoma , Oncolytic Viruses , RNA Viruses , Humans , Oncolytic Viruses/genetics , Melanoma/therapy , Vemurafenib , Epigenomics , Interferons , RNAABSTRACT
SOX10 is a key regulator of melanoma progression and promotes a melanocytic/differentiated state. Here we identified melanoma cell lines lacking SOX10 expression which retain their in vivo growth capabilities. More importantly, we find that SOX10 can regulate T-cell infiltration in melanoma while also decreasing common cancer stem cell (CSC) properties. We show that SOX10 regulates CEACAM1, a surface protein with immunomodulatory properties. SOX10 directly binds to a distal CEACAM1 promoter region approximately 3-4kbps from the CEACAM1 transcriptional start site. Furthermore, we show that a SOX10-CEACAM1 axis can suppress CD8+ T-cell infiltration as well as reduce CSC pool within tumors, leading to reduced tumor growth. Overall, these results identify SOX10 as a direct regulator of CEACAM1, and uncover both a pro- and anti-tumorigenic roles for SOX10 in melanoma.
ABSTRACT
Duchenne's muscular dystrophy (DMD) is a severe muscle wasting disorder characterized by the loss of dystrophin expression, muscle necrosis, inflammation and fibrosis. Ongoing muscle regeneration is impaired by persistent cytokine stress, further decreasing muscle function. Patients with DMD rarely survive beyond their early 20s, with cardiac and respiratory dysfunction being the primary cause of death. Despite an increase in our understanding of disease progression as well as promising preclinical animal models for therapeutic intervention, treatment options for muscular dystrophy remain limited and novel therapeutic targets are required. Many reports suggest that the TGFß signalling pathway is activated in dystrophic muscle and contributes to the pathology of DMD in part by impairing the differentiation of myoblasts into mature myofibers. Here, we show that in vitro knockdown of the Ste20-like kinase, SLK, can partially restore myoblast differentiation downstream of TGFß in a Smad2/3 independent manner. In an mdx model, we demonstrate that SLK is expressed at high levels in regenerating myofibers. Muscle-specific deletion of SLK reduced leukocyte infiltration, increased myogenin and utrophin expression and enhanced differentiation. This was accompanied by resistance to eccentric contraction-induced injury in slow fiber type-enriched soleus muscles. Finally, we found that these effects were partially dependent on the upregulation of p38 signalling. Collectively, these results demonstrate that SLK downregulation can restore some aspects of disease progression in DMD.
Subject(s)
Gene Knockout Techniques , MAP Kinase Signaling System/genetics , Muscle Development/genetics , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/metabolism , Protein Serine-Threonine Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Dogs , Mice , Mice, Inbred mdx , Mice, Knockout , Muscular Dystrophy, Duchenne/pathology , Myoblasts/metabolism , Myogenin/metabolism , Protein Serine-Threonine Kinases/genetics , Transforming Growth Factor beta/metabolismABSTRACT
The antimicrobial medication malarone (atovaquone/proguanil) is used as a fixed-dose combination for treating children and adults with uncomplicated malaria or as chemoprophylaxis for preventing malaria in travelers. It is an inexpensive, efficacious, and safe drug frequently prescribed around the world. Following anecdotal evidence from 17 patients in the provinces of Quebec and Ontario, Canada, suggesting that malarone/atovaquone may present some benefits in protecting against COVID-19, we sought to examine its antiviral potential in limiting the replication of SARS-CoV-2 in cellular models of infection. In VeroE6 expressing human TMPRSS2 and human lung Calu-3 epithelial cells, we show that the active compound atovaquone at micromolar concentrations potently inhibits the replication of SARS-CoV-2 and other variants of concern including the alpha, beta, and delta variants. Importantly, atovaquone retained its full antiviral activity in a primary human airway epithelium cell culture model. Mechanistically, we demonstrate that the atovaquone antiviral activity against SARS-CoV-2 is partially dependent on the expression of TMPRSS2 and that the drug can disrupt the interaction of the spike protein with the viral receptor, ACE2. Additionally, spike-mediated membrane fusion was also reduced in the presence of atovaquone. In the United States, two clinical trials of atovaquone administered alone or in combination with azithromycin were initiated in 2020. While we await the results of these trials, our findings in cellular infection models demonstrate that atovaquone is a potent antiviral FDA-approved drug against SARS-CoV-2 and other variants of concern in vitro.
Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Atovaquone/pharmacology , Humans , United StatesABSTRACT
HER2 is overexpressed in 20-30% of all breast cancers and is associated with an invasive disease and poor clinical outcome. The Ste20-like kinase (SLK) is activated downstream of HER2/Neu and is required for efficient epithelial-to-mesenchymal transition, cell cycle progression, and migration in the mammary epithelium. Here we show that loss of SLK in a murine model of HER2/Neu-positive breast cancers significantly accelerates tumor onset and decreases overall survival. Transcriptional profiling of SLK knockout HER2/Neu-derived tumor cells revealed a strong induction in the triple-negative breast cancer marker, Sox10, accompanied by an increase in mammary stem/progenitor activity. Similarly, we demonstrate that SLK and Sox10 expression are inversely correlated in patient samples, with the loss of SLK and acquisition of Sox10 marking the triple-negative subtype. Furthermore, pharmacological inhibition of AKT reduces SLK-null tumor growth in vivo and is rescued by ectopic Sox10 expression, suggesting that Sox10 is a critical regulator of tumor growth downstream of SLK/AKT. These findings highlight a role for SLK in negatively regulating HER2-induced mammary tumorigenesis and provide mechanistic insight into the regulation of Sox10 expression in breast cancer.
Subject(s)
Cell Transformation, Neoplastic/pathology , Protein Serine-Threonine Kinases/genetics , Receptor, ErbB-2/metabolism , SOXE Transcription Factors/genetics , Triple Negative Breast Neoplasms/pathology , Animals , Epithelial-Mesenchymal Transition/genetics , Female , Mice , Mice, SCID , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Spheroids, Cellular , Triple Negative Breast Neoplasms/genetics , Tumor Cells, CulturedABSTRACT
We report here a practical, enantioselective synthesis of benzofuran-derived, cyclic trans-beta-amino acid scaffold. In two cases, tricyclic derivatives having six- and eight-membered unsaturated lactams were obtained from this versatile scaffold. To explore the biological applications, these compounds were subjected to cell-based assays, using NIH3T3 mouse cells to examine their potency as cell motility inhibitors and identified 18 as a potent cell motility inhibitor (IC50 approximately 40 microM in chamber cell migration assay).
Subject(s)
Amino Acids, Cyclic/chemistry , Benzofurans/chemistry , Cell Movement/drug effects , Flavonoids/chemistry , Animals , Flavonoids/pharmacology , Mice , Molecular Probes , NIH 3T3 CellsABSTRACT
We have previously shown that the Ste20-like kinase SLK interacts directly with the LIM domain-binding protein 1, Ldb1. Ldb1 knock down in murine fibroblasts activates SLK and enhances cell migration. To investigate the effect of Ldb1 deletion in ErbB2/HER2-driven tumorigenesis, Ldb1 conditional mice were crossed into MMTV-NIC mice, expressing the Neu oncogene and Cre recombinase from a bi-cistronic transgene. Our results show that Ldb1 is expressed in the mammary epithelium and that deletion of Ldb1 does not impair mammary gland development. Although high levels of Ldb1 can be correlated with poor prognosis in HER2+ breast cancers, Ldb1 ablation does not affect Neu-induced tumor progression in transgenic mice. Surprisingly, Ldb1 deletion did not affect SLK kinase activity in primary tumors or established cell lines. Nevertheless, Ldb1-deficient tumor cells showed enhanced mesenchymal and migratory characteristics in vitro. However, Ldb1-null cells failed to colonize the lungs of wildtype female mice when injected into the tail vein. Together our results show that Ldb1 is dispensable for mammary gland development and Neu-induced tumor progression but required for dissemination at secondary sites. Furthermore, our data also highlight contrasting cell line behaviours observed from in vivo and in vitro assays.
Subject(s)
Cell Transformation, Neoplastic/genetics , DNA-Binding Proteins/genetics , LIM Domain Proteins/genetics , Receptor, ErbB-2/genetics , Animals , Cell Line , Cell Transformation, Neoplastic/metabolism , DNA-Binding Proteins/metabolism , Disease Models, Animal , Epithelial-Mesenchymal Transition , Gene Targeting , Heterografts , LIM Domain Proteins/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Serine-Threonine Kinases/genetics , Receptor, ErbB-2/metabolismABSTRACT
The Androgen Receptor (AR) has recently garnered a lot of attention as a potential biomarker and therapeutic target in hormone-dependent cancers, including breast cancer. However, several inconsistencies exist within the literature as to which subtypes of breast cancer express AR or whether it can be used to define its own unique subtype. Here, we analyze 1246 invasive breast cancer samples from the Cancer Genome Atlas and show that human breast cancers that have been subtyped based on their HER2, ESR1, or PGR expression contain four clusters of genes that are differentially expressed across all subtypes. We demonstrate that Sox10 is highly expressed in approximately one-third of all HER2/ESR1/PGR-low tumors and is a candidate biomarker of the triple-negative subtype. Although AR expression is acquired in many breast cancer cases, its expression could not define a unique subtype. Despite several reports stating that AR expression is acquired in HER2/ESR1/PGR triple-negative cancers, here we show that a low percentage of these cancers express AR (~20%). In contrast, AR is highly expressed in HER2-positive or ESR1/PGR-positive cancers (> 95%). Although AR expression cannot be used as an independent subtype biomarker, our analysis shows that routine evaluation of AR expression in tumors which express HER2, ESR1 and/or PGR may identify a unique subset of tumors which would benefit from anti-androgen based therapies.
ABSTRACT
Cortical spreading depression (CSD) induces waves of neuronal depolarization that confer neuroprotection to subsequent ischemic events in the rat brain. To gain insights into the molecular mechanisms elicited by CSD, we used representational difference analysis (RDA) to identify mRNAs induced by potassium depolarization in vivo. Using this approach, we have isolated a cDNA encoding the SIM2-related bHLH-PAS protein Nxf. Our results confirm that Nxf mRNA and protein are rapidly and transiently expressed in cortical neurons following CSD. Reporter assays show that Nxf is a transcriptional activator that associates with the bHLH-PAS sub-class co-factor ARNT2. Adenovirus-mediated expression of epitope-tagged Nxf results in cell death and the direct activation of the Bax gene in cultured cells. However, RNA interference studies show that endogenous Nxf is required for optimal neuroprotection by preconditioning in cultured F-11 cells. Together, our data indicate that Nxf is a novel bHLH-PAS transactivator transiently induced by preconditioning and that its sustained expression is detrimental. The identification of Nxf may represent an important step in our understanding of the molecular mechanisms of brain preconditioning and injury.