Insulin-like growth factor 1 signaling is essential for mitochondrial biogenesis and mitophagy in cancer cells.

Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator-activated receptor γ coactivator 1ß (PGC-1ß) and PGC-1α-related coactivator (PRC). Suppression of PGC-1ß and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1ß, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.

Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.

Direct interaction between scaffolding proteins RACK1 and 14-3-3ζ regulates brain-derived neurotrophic factor (BDNF) transcription.

RACK1 is a scaffolding protein that spatially and temporally regulates numerous signaling cascades. We previously found that activation of the cAMP signaling pathway induces the translocation of RACK1 to the nucleus. We further showed that nuclear RACK1 is required to promote the transcription of the brain-derived neurotrophic factor (BDNF). Here, we set out to elucidate the mechanism underlying cAMP-dependent RACK1 nuclear translocation and BDNF transcription. We identified the scaffolding protein 14-3-3ζ as a direct binding partner of RACK1. Moreover, we found that 14-3-3ζ was necessary for the cAMP-dependent translocation of RACK1 to the nucleus. We further observed that the disruption of RACK1/14-3-3ζ interaction with a peptide derived from the RACK1/14-3-3ζ binding site or shRNA-mediated 14-3-3ζ knockdown inhibited cAMP induction of BDNF transcription. Together, these data reveal that the function of nuclear RACK1 is mediated through its interaction with 14-3-3ζ. As RACK1 and 14-3-3ζ are two multifunctional scaffolding proteins that coordinate a wide variety of signaling events, their interaction is likely to regulate other essential cellular functions.

Serine phosphorylation of the insulin-like growth factor I (IGF-1) receptor C-terminal tail restrains kinase activity and cell growth.

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3ß in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3ß knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the (1248)SFYYS(1252) motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3ß restrains kinase activity and regulates receptor trafficking and signaling.

Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris.

RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris, RpfG together with the sensor kinase RpfC regulates multiple factors as a response to the cell-to-cell Diffusible Signalling Factor (DSF). A dynamic physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. Here we show that, contrary to expectation, regulation of motility by the GGDEF domain proteins does not depend upon their cyclic di-GMP synthetic activity. Furthermore we show that the complex of RpfG and GGDEF domain proteins recruits a specific PilZ domain 'adaptor' protein, and this complex then interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signalling influences motility through the highly regulated dynamic interaction of proteins that affect pilus action. A specific motif that we identify to be required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions is of broad relevance.

RACK1 to the future--a historical perspective.

This perspective summarises the first and long overdue RACK1 meeting held at the University of Limerick, Ireland, May 2013, in which RACK1's role in the immune system, the heart and the brain were discussed and its contribution to disease states such as cancer, cardiac hypertrophy and addiction were described. RACK1 is a scaffolding protein and a member of the WD repeat family of proteins. These proteins have a unique architectural assembly that facilitates protein anchoring and the stabilisation of protein activity. A large body of evidence is accumulating which is helping to define the versatile role of RACK1 in assembling and dismantling complex signaling pathways from the cell membrane to the nucleus in health and disease. In this commentary, we first provide a historical perspective on RACK1. We also address many of the pertinent and topical questions about this protein such as its role in transcription, epigenetics and translation, its cytoskeletal contribution and the merits of targeting RACK1 in disease.

PDLIM2 is highly expressed in Breast Cancer tumour-associated macrophages and is required for M2 macrophage polarization.

The PDZ-LIM domain-containing protein 2 (PDLIM2) regulates cell polarity and the protein stability of key transcription factors in epithelial and hemopoietic cells. We previously reported that PDLIM2 is more highly expressed in Triple Negative Breast Cancer (TNBC) than in other breast cancer types or normal breast tissue. In the course of the TNBC study, it was noted that PDLIM2 was highly expressed in the stroma of PDLIM2-expressing tumours. Here, we investigated the phenotype of these stromal cells and whether any infiltrating immune population was linked to PDLIM2 expression. We found that high PDLIM2 expression in breast tumours was associated with higher levels of infiltrating M2 macrophages, but was not associated with infiltrating T cell sub-populations. We then tested whether PDLIM2 contributes to macrophage differentiation or function by using cultures of bone marrow-derived macrophages from wildtype and Pdlim2 knockout mice. This demonstrated that PDLIM2 is required for naïve macrophage migration and for the full adoption of IL-4-induced M2 polarization, including expression of M2 phenotypic markers, cell adhesion and cell migration. TLR4-, TLR3- or IFNγ-induced M1 macrophage activity was less dependent on PDLIM2. Finally, analysis of publicly available breast cancer datasets showed that high PDLIM2 expression is associated with increased M2 macrophage infiltration. We conclude that PDLIM2 expression influences the tumour associated stroma and, in particular, M2 macrophage infiltration that may contribute to the progression of TNBC or other subsets of breast cancer.

Ribosomal 18 S RNA processing by the IGF-I-responsive WDR3 protein is integrated with p53 function in cancer cell proliferation.

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G(1) phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

Heme-binding protein HRG-1 is induced by insulin-like growth factor I and associates with the vacuolar H+-ATPase to control endosomal pH and receptor trafficking.

Endocytosis and trafficking of receptors and nutrient transporters are dependent on an acidic intra-endosomal pH that is maintained by the vacuolar H(+)-ATPase (V-ATPase) proton pump. V-ATPase activity has also been associated with cancer invasiveness. Here, we report on a new V-ATPase-associated protein, which we identified in insulin-like growth factor I (IGF-I) receptor-transformed cells, and which was separately identified in Caenorhabditis elegans as HRG-1, a member of a family of heme-regulated genes. We found that HRG-1 is present in endosomes but not in lysosomes, and it is trafficked to the plasma membrane upon nutrient withdrawal in mammalian cells. Suppression of HRG-1 with small interfering RNA causes impaired endocytosis of transferrin receptor, decreased cell motility, and decreased viability of HeLa cells. HRG-1 interacts with the c subunit of the V-ATPase and enhances V-ATPase activity in isolated yeast vacuoles. Endosomal acidity and V-ATPase assembly are decreased in cells with suppressed HRG-1, whereas transferrin receptor endocytosis is enhanced in cells that overexpress HRG-1. Cellular uptake of a fluorescent heme analogue is enhanced by HRG-1 in a V-ATPase-dependent manner. Our findings indicate that HRG-1 regulates V-ATPase activity, which is essential for endosomal acidification, heme binding, and receptor trafficking in mammalian cells. Thus, HRG-1 may facilitate tumor growth and cancer progression.

Tim-4 inhibition of T-cell activation and T helper type 17 differentiation requires both the immunoglobulin V and mucin domains and occurs via the mitogen-activated protein kinase pathway.

Emerging experimental data suggest an important role for the T-cell immunoglobulin mucin 1 (Tim-1):Tim-4 pathway in autoimmune and alloimmune responses in vivo. Using a Tim-4 ectodomain human IgG Fc fusion protein we studied the role of Tim-4 in T-cell activation, signalling and differentiation responses in vitro. We demonstrate that Tim-4Fc can inhibit naive and pre-activated T-cell activation, proliferation and cytokine secretion via a Tim-1-independent pathway. Tim-4 contains immunoglobulin variable (IgV) and mucin domains; to identify which domain accounts for the inhibitory effect novel Tim-4 fusion proteins containing either the IgV or mucin domain were generated. We demonstrate that both IgV and mucin domains are required for the inhibitory effects and that they are mediated at least in part by inhibition of extracellular signal-regulated kinase pathway activity. Given the emerging interest in the role of the Tim family in T helper type 17 (Th17) cells, which play an important role in autoimmune disease and transplantation tolerance, our data show that Tim-4Fc can prevent polarization of CD4(+) T cells to the Th17 phenotype. Collectively, our results highlight an inhibitory role for Tim-4Fc in vitro, which we propose is mediated by a receptor other than Tim-1. In addition, this study provides new insights into the role of Tim-4Fc in regulating Th17 immune responses and may open a new avenue for autoimmune therapy.

A case report of spontaneous mutation (C33>U) in the iron-responsive element of L-ferritin causing hyperferritinemia-cataract syndrome.

The hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disorder characterized by juvenile-onset cataracts and elevated serum ferritin levels. It is caused by mutation in the iron response element (IRE) within the 5'UTR of L-ferritin gene. The mutation results in a loss of post-transcriptional negative feedback exerted by the interaction between iron regulatory proteins 1, 2 (IRP1 and IRP2) and IRE, which leads to uncontrolled expression of L-ferritin. In this paper, we describe the molecular pathogenesis of non-hereditary hyperferritinemia cataract syndrome (non-H-HCS) in a patient with typical HHCS ocular lens morphology and high ferritin levels without obvious family history. Initial sequencing of the full-length L-ferritin cloned from genomic DNA demonstrated a mutation (C33>T) in the IRE of the affected patient but not in her unaffected family members. The mutation (C/T heterozygote) was also detected in cDNA derived from her blood mononuclear cells. Structure-prediction-modeling indicates that this mutation would significantly alter the secondary structure of the IRE, resulting in a loss of the interaction between IRP and IRE. By using IRP1/IRP2-human IgG1 Fc fusion proteins, we established a novel in vitro report system (modified ELISA) to verify impaired IRE/IRP binding. Both the C33>U and A40G mutations (the first identified mutation for HHCS) showed a dramatically decreased binding to IRP1/IRP2 protein, compared to the normal IRE RNA. Surprisingly, a decrease in L-ferritin mRNA levels was observed in the affected patient compared to controls suggesting a mechanism of transcriptional negative feedback by high intracellular L-ferritin protein levels not described heretofore. Taken together, spontaneous mutation in the IRE of L-ferritin may cause non-H-HCS by the same mechanism as HHCS. In addition, under abnormal circumstances, the protein level of L-ferritin may be principally controlled by post-transcriptional regulation rather than the transcriptional regulation. The successful establishment of an ELISA report system provides an alternative method to evaluate precisely the interaction between protein and RNA.

The insulin-like growth factor-I-mTOR signaling pathway induces the mitochondrial pyrimidine nucleotide carrier to promote cell growth.

The insulin/insulin-like growth factor (IGF) signaling pathway to mTOR is essential for the survival and growth of normal cells and also contributes to the genesis and progression of cancer. This signaling pathway is linked with regulation of mitochondrial function, but how is incompletely understood. Here we show that IGF-I and insulin induce rapid transcription of the mitochondrial pyrimidine nucleotide carrier PNC1, which shares significant identity with the essential yeast mitochondrial carrier Rim2p. PNC1 expression is dependent on PI-3 kinase and mTOR activity and is higher in transformed fibroblasts, cancer cell lines, and primary prostate cancers than in normal tissues. Overexpression of PNC1 enhances cell size, whereas suppression of PNC1 expression causes reduced cell size and retarded cell cycle progression and proliferation. Cells with reduced PNC1 expression have reduced mitochondrial UTP levels, but while mitochondrial membrane potential and cellular ATP are not altered, cellular ROS levels are increased. Overall the data indicate that PNC1 is a target of the IGF-I/mTOR pathway that is essential for mitochondrial activity in regulating cell growth and proliferation.

Controlled Signaling-Insulin-Like Growth Factor Receptor Endocytosis and Presence at Intracellular Compartments.

Ligand-induced activation of the IGF-1 receptor triggers plasma-membrane-derived signal transduction but also triggers receptor endocytosis, which was previously thought to limit signaling. However, it is becoming ever more clear that IGF-1R endocytosis and trafficking to specific subcellular locations can define specific signaling responses that are important for key biological processes in normal cells and cancer cells. In different cell types, specific cell adhesion receptors and associated proteins can regulate IGF-1R endocytosis and trafficking. Once internalized, the IGF-1R may be recycled, degraded or translocated to the intracellular membrane compartments of the Golgi apparatus or the nucleus. The IGF-1R is present in the Golgi apparatus of migratory cancer cells where its signaling contributes to aggressive cancer behaviors including cell migration. The IGF-1R is also found in the nucleus of certain cancer cells where it can regulate gene expression. Nuclear IGF-1R is associated with poor clinical outcomes. IGF-1R signaling has also been shown to support mitochondrial biogenesis and function, and IGF-1R inhibition causes mitochondrial dysfunction. How IGF-1R intracellular trafficking and compartmentalized signaling is controlled is still unknown. This is an important area for further study, particularly in cancer.

IGF-1 Signalling Regulates Mitochondria Dynamics and Turnover through a Conserved GSK-3ß-Nrf2-BNIP3 Pathway.

The Insulin-like Growth Factor I (IGF-1) signalling pathway is essential for cell growth and facilitates tumourogenic processes. We recently reported that IGF-1 induces a transcriptional programme for mitochondrial biogenesis, while also inducing expression of the mitophagy receptor BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), suggesting that IGF-1 has a key mitochondria-protective role in cancer cells. Here, we investigated this further and delineated the signaling pathway for BNIP3 induction. We established that IGF-1 induced BNIP3 expression through a known AKT serine/threonine kinase 1 (AKT)-mediated inhibitory phosphorylation on Glycogen Synthase Kinase-3ß (GSK-3ß), leading to activation of Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2/Nrf2) and acting through the downstream transcriptional regulators Nuclear Respiratory Factor-1 (NRF1) and Hypoxia-inducible Factor 1 subunit α (HIF-1α). Suppression of IGF-1 signaling, Nrf2 or BNIP3 caused the accumulation of elongated mitochondria and altered the mitochondrial dynamics. IGF-1R null Mouse Embryonic Fibroblasts (MEFs) were impaired in the BNIP3 expression and in the capacity to mount a cell survival response in response to serum deprivation or mitochondrial stress. IGF-1 signalling enhanced the cellular capacity to induce autophagosomal turnover in response to activation of either general autophagy or mitophagy. Overall, we conclude that IGF-1 mediated a mitochondria-protective signal that was coordinated through the cytoprotective transcription factor Nrf2. This pathway coupled mitochondrial biogenesis with BNIP3 induction, and increased the cellular capacity for autophagosome turnover, whilst enhancing survival under conditions of metabolic or mitochondrial stress.

IGF-1 receptor activity in the Golgi of migratory cancer cells depends on adhesion-dependent phosphorylation of Tyr1250 and Tyr1251.

Although insulin-like growth factor 1 (IGF-1) signaling promotes tumor growth and cancer progression, therapies that target the IGF-1 receptor (IGF-1R) have shown poor clinical efficacy. To address IGF-1R activity in cancer cells and how it differs from that of the closely related insulin receptor (IR), we focused on two tyrosines in the IGF-1R C-terminal tail that are not present in the IR and are essential for IGF-1-mediated cancer cell survival, migration, and tumorigenic growth. We found that Tyr1250 and Tyr1251 (Tyr1250/1251) were autophosphorylated in a cell adhesion-dependent manner. To investigate the consequences of this phosphorylation, we generated phosphomimetic Y1250E/Y1251E (EE) and nonphosphorylatable Y1250F/Y1251F (FF) mutant forms of IGF-1R. Although fully competent in kinase activity and signaling, the EE mutant was more rapidly internalized and degraded than either the wild-type or FF receptor. IGF-1 promoted the accumulation of wild-type and EE IGF-1R within the Golgi apparatus, whereas the FF mutant remained at the plasma membrane. Golgi-associated IGF-1R signaling was a feature of migratory cancer cells, and Golgi disruption impaired IGF-1-induced signaling and cell migration. Upon the formation of new cell adhesions, IGF-1R transiently relocalized to the plasma membrane from the Golgi. Thus, phosphorylation at Tyr1250/1251 promoted IGF-1R translocation to and signaling from the Golgi to support an aggressive cancer phenotype. This process distinguishes IGF-1R from IR signaling and could contribute to the poor clinical efficacy of antibodies that target IGF-1R on the cell surface.

Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and beta1 integrin to promote cell migration.

The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit beta1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and beta1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either beta1 integrin or PP2A in a complex at the IGF-IR.

A Novel Role for Cathepsin S as a Potential Biomarker in Triple Negative Breast Cancer.

Cathepsin S (CTSS) has previously been implicated in a number of cancer types, where it is associated with poor clinical features and outcome. To date, patient outcome in breast cancer has not been examined with respect to this protease. Here, we carried out immunohistochemical (IHC) staining of CTSS using a breast cancer tissue microarray in patients who received adjuvant therapy. We scored CTSS expression in the epithelial and stromal compartments and evaluated the association of CTSS expression with matched clinical outcome data. We observed differences in outcome based on CTSS expression, with stromal-derived CTSS expression correlating with a poor outcome and epithelial CTSS expression associated with an improved outcome. Further subtype characterisation revealed high epithelial CTSS expression in TNBC patients with improved outcome, which remained consistent across two independent TMA cohorts. Further in silico gene expression analysis, using both in-house and publicly available datasets, confirmed these observations and suggested high CTSS expression may also be beneficial to outcome in ER-/HER2+ cancer. Furthermore, high CTSS expression was associated with the BL1 Lehmann subgroup, which is characterised by defects in DNA damage repair pathways and correlates with improved outcome. Finally, analysis of matching IHC analysis reveals an increased M1 (tumour destructive) polarisation in macrophage in patients exhibiting high epithelial CTSS expression. In conclusion, our observations suggest epithelial CTSS expression may be prognostic of improved outcome in TNBC. Improved outcome observed with HER2+ at the gene expression level furthermore suggests CTSS may be prognostic of improved outcome in ER- cancers as a whole. Lastly, from the context of these patients receiving adjuvant therapy and as a result of its association with BL1 subgroup CTSS may be elevated in patients with defects in DNA damage repair pathways, indicating it may be predictive of tumour sensitivity to DNA damaging agents.

PDLIM2 Is a Marker of Adhesion and ß-Catenin Activity in Triple-Negative Breast Cancer.

The PDLIM2 protein regulates stability of transcription factors including NF-κB and STATs in epithelial and hemopoietic cells. PDLIM2 is strongly expressed in certain cancer cell lines that exhibit an epithelial-to-mesenchymal phenotype, and its suppression is sufficient to reverse this phenotype. PDLIM2 supports the epithelial polarity of nontransformed breast cells, suggesting distinct roles in tumor suppression and oncogenesis. To better understand its overall function, we investigated PDLIM2 expression and activity in breast cancer. PDLIM2 protein was present in 60% of tumors diagnosed as triple-negative breast cancer (TNBC), and only 20% of other breast cancer subtypes. High PDLIM2 expression in TNBC was positively correlated with adhesion signaling and ß-catenin activity. Interestingly, PDLIM2 was restricted to the cytoplasm/membrane of TNBC cells and excluded from the nucleus. In breast cell lines, PDLIM2 retention in the cytoplasm was controlled by cell adhesion, and translocation to the nucleus was stimulated by insulin-like growth factor-1 or TGFß. Cytoplasmic PDLIM2 was associated with active ß-catenin and ectopic expression of PDLIM2 was sufficient to increase ß-catenin levels and its transcriptional activity in reporter assays. Suppression of PDLIM2 inhibited tumor growth in vivo, whereas overexpression of PDLIM2 disrupted growth in 3D cultures. These results suggest that PDLIM2 may serve as a predictive biomarker for a large subset of TNBC whose phenotype depends on adhesion-regulated ß-catenin activity and which may be amenable to therapies that target these pathways. SIGNIFICANCE: This study shows that PDLIM2 expression defines a subset of triple-negative breast cancer that may benefit from targeting the ß-catenin and adhesion signaling pathways. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2619/F1.large.jpg.

The EphA3 receptor is expressed in a subset of rhabdomyosarcoma cell lines and suppresses cell adhesion and migration.

Elevated expression of the Eph receptor tyrosine kinase EphA3 is associated with lymphocytic leukaemia, but little is known about its expression or function in solid tumours. Out of a panel of cancer cell lines, we found that EphA3 was expressed only on two rhabdomyosarcoma (RMS) cell lines of the embryonal histological subtype and on one of the alveolar RMS subtype, whereas it was not detected on two other cell lines of the alveolar subtype. Other EphA receptors (1-7) were, either not expressed in any, or expressed in all five RMS cell lines. Stimulation of EphA3-expressing TE671 and RD RMS cells with ephrinA5 resulted in loss of adhesion to fibronectin, decreased migration towards the stromal cell-derived growth factor-I (SDF-I), increased EphA3 phosphorylation, and increased Rho GTPase activity. In contrast, ectopic expression of EphA3 in the EphA3 negative CRL2061 cell line resulted in decreased cell adhesion. Finally, suppression of EphA3 expression by siRNA in RD cells results in increased SDF-I-mediated motility. These data indicate that EphA3 expression may define subsets of RMS tumours, and that EphA3 suppresses motility through regulation of Rho GTPases in RMS cells.

Metastatic susceptibility locus, an 8p hot-spot for tumour progression disrupted in colorectal liver metastases: 13 candidate genes examined at the DNA, mRNA and protein level.

BACKGROUND: Mortality from colorectal cancer is mainly due to metastatic liver disease. Improved understanding of the molecular events underlying metastasis is crucial for the development of new methods for early detection and treatment of colorectal cancer. Loss of chromosome 8p is frequently seen in colorectal cancer and implicated in later stage disease and metastasis, although a single metastasis suppressor gene has yet to be identified. We therefore examined 8p for genes involved in colorectal cancer progression. METHODS: Loss of heterozygosity analyses were used to map genetic loss in colorectal liver metastases. Candidate genes in the region of loss were investigated in clinical samples from 44 patients, including 6 with matched colon normal, colon tumour and liver metastasis. We investigated gene disruption at the level of DNA, mRNA and protein using a combination of mutation, semi-quantitative real-time PCR, western blotting and immunohistochemical analyses. RESULTS: We mapped a 2 Mb region of 8p21-22 with loss of heterozygosity in 73% of samples; 8/11 liver metastasis samples had loss which was not present in the corresponding matched primary colon tumour. 13 candidate genes were identified for further analysis. Both up and down-regulation of 8p21-22 gene expression was associated with metastasis. ADAMDEC1 mRNA and protein expression decreased during both tumourigenesis and tumour progression. Increased STC1 and LOXL2 mRNA expression occurred during tumourigenesis. Liver metastases with low DcR1/TNFRSF10C mRNA expression were more likely to present with extrahepatic metastases (p = 0.005). A novel germline truncating mutation of DR5/TNFRSF10B was identified, and DR4/TNFRSF10A SNP rs4872077 was associated with the development of liver metastases (p = 0.02). CONCLUSION: Our data confirm that genes on 8p21-22 are dysregulated during colorectal cancer progression. Interestingly, however, instead of harbouring a single candidate colorectal metastasis suppressor 8p21-22 appears to be a hot-spot for tumour progression, encoding at least 13 genes with a putative role in carcinoma development. Thus, we propose that this region of 8p comprises a metastatic susceptibility locus involved in tumour progression whose disruption increases metastatic potential.