Preclinical Efficacy of Cystatin C to Target the Oncogenic Activity of Transforming Growth Factor β in Breast Cancer1,2

We previously identified cystatin C (CystC) as a novel antagonist of transforming growth factor β (TGF-β) signaling in normal and malignant cells. However, whether the anti-TGF-β activities of CystC can be translated to preclinical animal models of breast cancer growth and metastasis remains unproven. Assessing the preclinical efficacy of CystC was accomplished using metastatic 4T1 breast cancer cells, whose oncogenic responses to TGF-β were inhibited both in vitro and in vivo. Indeed, we observed CystC to prevent TGF-β from stimulating the growth and pulmonary metastasis of 4T1 tumors in mice in part by reducing the extent of Smad2, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 phosphorylation present in 4T1 tumors. We also found CystC to significantly antagonize angiogenesis in developing 4T1 tumors, suggesting a novel role for CystC in uncoupling TGF-β signaling in endothelial cells (ECs). Accordingly, CystC dramatically reduced murine and human EC responsiveness to TGF-β, including their ability to regulate the expression of 1) TGF-β signaling components, 2) inhibitor of differentiation (ID) family members, and 3) matrix metalloproteinases and their inhibitors (TIMPs) and to undergo cell invasion and angiogenic sprouting stimulated by TGF-β. Importantly, CystC prevented TGF-β from stimulating vessel development in Matrigel plugs implanted into genetically normal mice. Collectively, our findings provide the first preclinical evidence that CystC is efficacious in preventing breast cancer progression and angiogenesis stimulated by the oncogenic TGF-β signaling system and suggest that CystC-based chemotherapeutics possesses translational efficacy to one day treat and improve the clinical course of late-stage breast cancers

PGE2 receptor EP2 mediates the antagonistic effect of COX-2 on TGF-β signaling during mammary tumorigenesis

The molecular mechanisms that enable cyclooxygenase-2 (COX-2) and its mediator prostaglandin E2 (PGE2) to inhibit transforming growth factor-β (TGF-β) signaling during mammary tumorigenesis remain unknown. We show here that TGF-β selectively stimulated the expression of the PGE2 receptor EP2, which increased normal and malignant mammary epithelial cell (MEC) invasion, anchorage-independent growth, and resistance to TGF-β-induced cytostasis. Mechanistically, elevated EP2 expression in normal MECs inhibited the coupling of TGF-β to Smad2/3 activation and plasminogen activator inhibitor-1 (PAI1) expression, while EP2 deficiency in these same MECs augmented Smad2/3 activation and PAI expression stimulated by TGF-β. Along these lines, engineering malignant MECs to lack EP2 expression prevented their growth in soft agar, restored their cytostatic response to TGF-β, decreased their invasiveness in response to TGF-β, and potentiated their activation of Smad2/3 and expression of PAI stimulated by TGF-β. More important, we show that COX-2 or EP2 deficiency both significantly decreased the growth, angiogenesis, and pulmonary metastasis of mammary tumors produced in mice. Collectively, this investigation establishes EP2 as a potent mediator of the anti-TGF-β activities elicited by COX-2/PGE2 in normal and malignant MECs. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the oncogenic activities of TGF-β during mammary tumorigenesis.—Tian, M., Schiemann, W. P. PGE2 receptor EP2 mediates the antagonistic effect of COX-2 on TGF-β signaling during mammary tumorigenesis

Deptor Enhances Triple-Negative Breast Cancer Metastasis and Chemoresistance through Coupling to Survivin Expression

Transforming growth factor–β (TGF-β) functions to suppress tumorigenesis in normal mammary tissues and early-stage breast cancers and, paradoxically, acts to promote the metastasis and chemoresistance in late-stage breast cancers, particularly triple-negative breast cancers (TNBCs). Precisely how TGF-β acquires oncogenic characteristics in late-stage breast cancers remains unknown, as does the role of the endogenous mammalian target of rapamycin (mTOR) inhibitor, Dep domain–containing mTOR-interacting protein (Deptor), in coupling TGF-β to TNBC development and metastatic progression. Here we demonstrate that Deptor expression was downregulated in basal-like/TNBCs relative to their luminal counterparts. Additionally, Deptor expression was 1) inversely correlated with the metastatic ability of human (MCF10A) and mouse (4T1) TNBC progression series and 2) robustly repressed by several inducers of epithelial-mesenchymal transition programs. Functional disruption of Deptor expression in 4T07 cells significantly inhibited their proliferation and organoid growth in vitro, as well as prevented their colonization and tumor formation in the lungs of mice. In stark contrast, elevated Deptor expression was significantly associated with poorer overall survival of patients harboring estrogen receptor α–negative breast cancers. Accordingly, enforced Deptor expression in MDA-MB-231 cells dramatically enhanced their 1) organoid growth in vitro, 2) pulmonary outgrowth in mice, and 3) resistance to chemotherapies, an event dependent on the coupling of Deptor to survivin expression. Collectively, our findings highlight the dichotomous functions of Deptor in modulating the proliferation and survival of TNBCs during metastasis; they also implicate Deptor and its stimulation of survivin as essential components of TNBC resistance to chemotherapies and apoptotic stimuli

NetSlim:High-confidence curated signaling maps

We previously developed NetPath as a resource for comprehensive manually curated signal transduction pathways. The pathways in NetPath contain a large number of molecules and reactions which can sometimes be difficult to visualize or interpret given their complexity. To overcome this potential limitation, we have developed a set of more stringent curation and inclusion criteria for pathway reactions to generate high-confidence signaling maps. NetSlim is a new resource that contains this ‘core’ subset of reactions for each pathway for easy visualization and manipulation. The pathways in NetSlim are freely available at http://www.netpath.org/netslim. Database URL: www.netpath.org/netsli

X-linked Inhibitor of Apoptosis Protein and Its E3 Ligase Activity Promote Transforming Growth Factor-β-mediated Nuclear Factor-κB 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