Jason R. Neil

Transforming Growth Factor-β and the Hallmarks of Cancer

Tumorigenesis is in many respects a process of dysregulated cellular evolution that drives malignant cells to acquire six phenotypic hallmarks of cancer, including their ability to proliferate and replicate autonomously, to resist cytostatic and apoptotic signals, and to induce tissue invasion, metastasis, and angiogenesis. Transforming growth factor-β (TGF-β) is a potent pleiotropic cytokine that functions as a formidable barrier to the development of cancer hallmarks in normal cells and tissues. Paradoxically, tumorigenesis counteracts the tumor suppressing activities of TGF-β, thus enabling TGF-β to stimulate cancer invasion and metastasis. Fundamental gaps exist in our knowledge of how malignant cells overcome the cytostatic actions of TGF-β, and of how TGF-β stimulates the acquisition of cancer hallmarks by developing and progressing human cancers. Here we review the molecular and cellular mechanisms that underlie the ability of TGF-β to mediate tumor suppression in normal cells, and conversely, to facilitate cancer progression and disease dissemination in malignant cells.

Fibulins 3 and 5 Antagonize Tumor Angiogenesis In vivo

Lethal tumor growth and progression cannot occur without angiogenesis, which facilitates cancer cell proliferation, survival, and dissemination. Fibulins (FBLN) 5 and 3 are widely expressed extracellular matrix proteins that regulate cell proliferation in a context-specific manner. Reduced FBLN-5 expression has been associated with cancer formation and progression in humans, whereas its constitutive expression antagonizes endothelial cell angiogenic sprouting in vitro. Thus, FBLN-5 may suppress tumorigenesis by preventing tumor angiogenesis. FBLN-3 is homologous to FBLN-5 and expressed in endothelial cells, yet its role in tumorigenesis and angiogenesis is unknown. We find FBLN-3 expression to be altered in some human tumors and that its constitutive expression in endothelial cells inhibited their proliferation, invasion, and angiogenic sprouting, as well as their response to vascular endothelial growth factor as measured by p38 mitogen-activated protein kinase activation. In endothelial cells, both FBLNs (a) reduced angiogenic sprouting stimulated by basic fibroblast growth factor (bFGF); (b) inhibited matrix metalloproteinase expression and activity; and (c) stimulated tissue inhibitor of metalloproteinase expression. More importantly, both FBLNs prevented angiogenesis and vessel infiltration into bFGF-supplemented Matrigel plugs implanted in genetically normal mice, as well as decreased the growth and blood vessel density in tumors produced by MCA102 fibrosarcoma cells implanted s.c. into syngeneic mice. Our findings establish FBLN-3 and FBLN-5 as novel angiostatic agents capable of reducing tumor angiogenesis and, consequently, tumor growth in vivo and suggest that these angiostatic activities may one day be exploited to combat tumor angiogenesis and metastasis in cancer patients.

Combination antibody treatment down-regulates epidermal growth factor receptor by inhibiting endosomal recycling

Due to its common dysregulation in epithelial-based cancers and extensive characterization of its role in tumor growth, epidermal growth factor receptor (EGFR) is a highly validated target for anticancer therapies. There has been particular interest in the development of monoclonal antibodies (mAbs) targeting EGFR, resulting in two approved mAb-based drugs and several others in clinical trials. It has recently been reported that treatment with combinations of noncompetitive mAbs can induce receptor clustering, leading to synergistic receptor down-regulation. We elucidate three key aspects of this phenomenon. First, we show that highly potent combinations consisting of two noncompetitive mAbs that target EGFR domain 3 reduce surface receptor levels by up to 80% with a halftime of 0.5–5 h in both normal and transformed human cell lines to an extent inversely proportional to receptor density. Second, we find the mechanism underlying down-regulation to be consistent with recycling inhibition. Third, in contrast to the agonism associated with ligand-induced down-regulation, we demonstrate that mAb-induced down-regulation does not activate EGFR or its downstream effectors and it leads to synergistic reduction in migration and proliferation of cells that secrete autocrine ligand. These new insights will aid in ongoing rational design of EGFR-targeted antibody therapeutics.

Cox-2 inactivates Smad signaling and enhances EMT stimulated by TGF-β through a PGE2-dependent mechanisms

Although it is well established that mammary tumorigenesis converts transforming growth factor-beta (TGF-beta) from a tumor suppressor to a tumor promoter, the molecular, cellular and microenvironmental mechanisms underlying the dichotomous nature of TGF-beta in mammary epithelial cells (MECs) remains to be determined definitively. Aberrant upregulation of the inducible cyclooxygenase, Cox-2, occurs frequently in breast cancers and is associated with increasing disease severity and the acquisition of metastasis; however, the impact of Cox-2 expression on normal and malignant MEC response to TGF-beta remains unknown. We show here that TGF-beta induced Cox-2 expression in normal MECs during their acquisition of an epithelial-mesenchymal transition (EMT) phenotype. Moreover, stable Cox-2 expression in normal MECs stimulated their invasion, EMT and anchorage-independent growth and inhibited their activation of Smad2/3 by TGF-beta. Conversely, antagonizing TGF-beta signaling in malignant, metastatic MECs significantly reduced their expression of Cox-2 as well as enhanced their activation of Smad2/3 by TGF-beta. Along these lines, elevated Cox-2 expression elicited prostaglandin E(2) (PGE(2)) production and the autocrine activation of EP receptors, which antagonized Smad2/3 signaling in normal and malignant MECs. Importantly, rendering normal and malignant MECs Cox-2 deficient inhibited their production of PGE(2) and acquisition of an EMT morphology as well as potentiated their nuclear accumulation of Smad2/3 and transcription of plasminogen activator inhibitor-1 and p15 messenger RNA. Collectively, our findings establish Cox-2 as a novel antagonist of Smad2/3 signaling in normal and malignant MECs; they also suggest that chemotherapeutic targeting of Cox-2 may offer new inroads in restoring the tumor-suppressing activities of TGF-beta in malignant, metastatic breast cancers.

The use of cystatin C to inhibit epithelial–mesenchymal transition and morphological transformation stimulated by transforming growth factor-β

IntroductionTransforming growth factor-β (TGF-β) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-β-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-β. Recent findings suggest that therapeutics designed to antagonize TGF-β signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-β type II receptor antagonist that inhibits TGF-β binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-β, particularly its stimulation of mammary epithelial–mesenchymal transition (EMT), can be prevented by CystC.MethodRetroviral infection was used to constitutively express CystC or a CystC mutant impaired in its ability to inhibit cathepsin protease activity (namely Δ14CystC) in murine NMuMG MECs and in normal rat kidney (NRK) fibroblasts. The effect of recombinant CystC administration or CystC expression on TGF-β stimulation of NMuMG cell EMT in vitro was determined with immunofluorescence to monitor rearrangements of actin cytoskeletal architecture and E-cadherin expression. Soft-agar growth assays were performed to determine the effectiveness of CystC in preventing TGF-β stimulation of morphological transformation and anchorage-independent growth in NRK fibroblasts. Matrigel invasion assays were performed to determine the ability of CystC to inhibit NMuMG and NRK motility stimulated by TGF-β.ResultsCystC and Δ14CystC both inhibited NMuMG cell EMT and invasion stimulated by TGF-β by preventing actin cytoskeletal rearrangements and E-cadherin downregulation. Moreover, both CystC molecules completely antagonized TGF-β-mediated morphological transformation and anchorage-independent growth of NRK cells, and inhibited their invasion through synthetic basement membranes. Both CystC and Δ14CystC also inhibited TGF-β signaling in two tumorigenic human breast cancer cell lines.ConclusionOur findings show that TGF-β stimulation of initiating metastatic events, including decreased cell polarization, reduced cell–cell contact, and elevated cell invasion and migration, are prevented by CystC treatment. Our findings also suggest that the future development of CystC or its peptide mimetics hold the potential to improve the therapeutic response of human breast cancers regulated by TGF-β.

X-linked inhibitor of apoptosis protein (xIAP) and its E3 ligase activity promotes transforming growth factor beta(TGF-beta;)-mediated nuclear factor-kappaB (NFkappaB) activation during breast cancer progression

The precise sequence of events that enable mammary tumorigenesis to convert transforming growth factor-β (TGF-β) from a tumor suppressor to a tumor promoter remains incompletely understood. We show here that X-linked inhibitor of apoptosis protein (xIAP) is essential for the ability of TGF-β to stimulate nuclear factor-κB (NF-κB) in metastatic 4T1 breast cancer cells. Indeed whereas TGF-β suppressed NF-κB activity in normal mammary epithelial cells, those engineered to overexpress xIAP demonstrated activation of NF-κB when stimulated with TGF-β. Additionally up-regulated xIAP expression also potentiated the basal and TGF-β-stimulated transcriptional activities of Smad2/3 and NF-κB. Mechanistically xIAP (i) interacted physically with the TGF-β type I receptor, (ii) mediated the ubiquitination of TGF-β-activated kinase 1 (TAK1), and (iii) facilitated the formation of complexes between TAK1-binding protein 1 (TAB1) and IκB kinase β that enabled TGF-β to activate p65/RelA and to induce the expression of prometastatic (i.e. cyclooxygenase-2 and plasminogen activator inhibitor-1) and prosurvival (i.e. survivin) genes. We further observed that inhibiting the E3 ubiquitin ligase function of xIAP or expressing a mutant ubiquitin protein (i.e. K63R-ubiquitin) was capable of blocking xIAP- and TGF-β-mediated activation of NF-κB. Functionally xIAP deficiency dramatically reduced the coupling of TGF-β to Smad2/3 in NMuMG cells as well as inhibited their expression of mesenchymal markers in response to TGF-β. More importantly, xIAP deficiency also abrogated the formation of TAB1·IκB kinase β complexes in 4T1 breast cancer cells, thereby diminishing their activation of NF-κB, their expression of prosurvival/metastatic genes, their invasion through synthetic basement membranes, and their growth in soft agar. Collectively our findings have defined a novel role for xIAP in mediating oncogenic signaling by TGF-β in breast cancer cells.

Epidermal growth factor receptor downregulation by small heterodimeric binding proteins

No single engineered protein has been shown previously to robustly downregulate epidermal growth factor receptor (EGFR), a validated cancer target. A panel of fibronectin-based domains was engineered to bind with picomolar to nanomolar affinity to multiple epitopes of EGFR. Monovalent and homo- and hetero-bivalent dimers of these domains were tested for EGFR downregulation. Selected orientations of non-competitive heterodimers decrease EGFR levels by up to 80% in multiple cell types, without activating receptor signaling. These heterodimers inhibit autophosphorylation, proliferation and migration, and are synergistic with the monoclonal antibody cetuximab in these activities. These small (25 kDa) heterodimers represent a novel modality for modulating surface receptor levels.

PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

The actin-binding protein Mena regulates RTK signaling after growth factor stimulation in tumor cells by a novel mechanism. The alternatively spliced MenaINV isoform disrupts this attenuation to drive sensitivity to growth factors, resistance to targeted inhibitors, and ultimately tumor invasion and metastasis.

Cancer Signaling Network Analysis by Quantitative Mass Spectrometry

Global analysis of protein phosphorylation by mass spectrometry (MS) provides unbiased, discovery-based, site-specific monitoring of phosphorylation sites governing cell signaling networks involved in cancer progression and therapeutic resistance. In this chapter, advances in MS instrumentation and methodology for the identification and quantification of protein phosphorylation are discussed. These topics include (1) advantages and limitations of current MS-based protocols, (2) fundamentals of phosphopeptide fragmentation and identification, (3) selection of MS instrumentation, (4) fractionation and enrichment methods for detecting phosphorylated proteins/peptides, and (5) methods for phosphoproteome quantification. The final two topics represent the most important subjects of this chapter, as fractionation, enrichment, and quantification are crucially important to the generation of high quality MS-based phosphoproteomic data. These sections detail the use of immunoaffinity enrichment, immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and strong cation exchange (SCX) chromatography as key methods for enriching and fractionating complex biological samples for phosphoproteomic analysis. Quantification of changes in signaling networks at the phosphoproteomic level through metabolic labeling (e.g., SILAC), chemical modification (e.g., iTRAQ), or label-free quantification is presented. As significant progress in detection and quantification strategies in phosphoproteomic research has arisen over the last decade, implementation of these approaches will enhance our understanding of cell signaling networks involved in cancer progression and thereby improve therapeutic targeting and monitoring of therapeutic efficacy.