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The neutrophil elastase inhibitor elafin triggers rb-mediated growth arrest and caspase-dependent apoptosis in breast cancer.

ABSTRACT: Elafin, an endogenous inhibitor of neutrophil elastase, is expressed in human mammary epithelial cells but is transcriptionally downregulated in breast cancer cells. We hypothesized that elafin may exert a tumor-suppressive activity in the context of breast cancer. In this study, we show that the retinoblastoma (Rb) pathway governs the antitumor properties of elafin. In breast cancer cells with functional Rb, the expression of elafin triggered Rb-dependent cell cycle arrest. Elafin also exhibited suppressive activity in breast cancer cell lines lacking Rb, but this was associated with an induction of caspase-3-dependent, p53-independent apoptotic cell death. Normal mammary epithelial cells were not affected by elafin. Collectively, these results argue that elafin mediates tumor-suppressive effects that are cytostatic or cytotoxic depending on the Rb status. Our findings suggest that elafin could be engineered as a therapeutic modality to treat breast cancer without toxicity to normal proliferating cells.

SUBMITTER: Caruso JA

PROVIDER: S-EPMC2940941 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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The neutrophil elastase inhibitor elafin triggers rb-mediated growth arrest and caspase-dependent apoptosis in breast cancer.

Caruso Joseph A JA Hunt Kelly K KK Keyomarsi Khandan K

Cancer research 20100907 18

Elafin, an endogenous inhibitor of neutrophil elastase, is expressed in human mammary epithelial cells but is transcriptionally downregulated in breast cancer cells. We hypothesized that elafin may exert a tumor-suppressive activity in the context of breast cancer. In this study, we show that the retinoblastoma (Rb) pathway governs the antitumor properties of elafin. In breast cancer cells with functional Rb, the expression of elafin triggered Rb-dependent cell cycle arrest. Elafin also exhibite ...[more]

PMID: 20823156

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Project description:The serine protease inhibitor, elafin, is a critical component of the epithelial barrier against neutrophil elastase (NE). Elafin is downregulated in the majority of breast cancer cell lines compared with normal human mammary epithelial cells (HMECs). Here, we evaluated the role of elafin and NE on proliferation and tumorigenesis. Elafin is induced in growth factor-deprived HMECs as they enter a quiescent (G0) state, suggesting that elafin is a counterbalance against the mitogenic effects of NE in G0 HMECs. Stable knockdown of elafin compromises the ability of HMECs to maintain G0 arrest during long-term growth factor deprivation; this effect can be reversed by re-expression of wild-type elafin but not elafin-M25G lacking protease inhibitory function. These results suggest that NE, which is largely contributed by activated neutrophils in the tumor microenvironment, may be negatively regulating the ability of elafin to arrest cells in G0. In fact when purified NE was added to elafin-knocked down HMECs, these cells demonstrated greater sensitivity to the growth-promoting effects of purified NE. Activation of ERK signaling, downstream of toll-like receptor 4, was essential to the mitogenic effect of NE on HMECs. These findings were next translated to patient samples. Immunohistochemical analysis of normal breast tissue revealed robust elafin expression in the mammary epithelium; however, elafin expression was dramatically downregulated in a significant proportion of human breast tumor specimens. The loss of elafin expression during breast cancer progression may promote tumor growth as a consequence of increased NE activity. To address the role of NE in mammary tumorigenesis, we next examined whether deregulated NE activity enhances mammary tumor growth. NE knockout in the C3(1)TAg mouse model of mammary tumorigenesis suppressed proliferation and reduced the kinetics of tumor growth. Overall, the imbalance between NE and its inhibitors, such as elafin, presents an important therapeutic target in breast cancer.

Project description:BackgroundPreclinical evidence implicates neutrophil elastase (NE) in pulmonary arterial hypertension (PAH) pathogenesis, and the NE inhibitor elafin is under early therapeutic investigation.Research questionAre circulating NE and elafin levels abnormal in PAH and are they associated with clinical severity?Study design and methodsIn an observational Stanford University PAH cohort (n = 249), plasma NE and elafin levels were measured in comparison with those of healthy control participants (n = 106). NE and elafin measurements were then related to PAH clinical features and relevant ancillary biomarkers. Cox regression models were fitted with cubic spline functions to associate NE and elafin levels with survival. To validate prognostic relationships, we analyzed two United Kingdom cohorts (n = 75 and n = 357). Mixed-effects models evaluated NE and elafin changes during disease progression. Finally, we studied effects of NE-elafin balance on pulmonary artery endothelial cells (PAECs) from patients with PAH.ResultsRelative to control participants, patients with PAH were found to have increased NE levels (205.1 ng/mL [interquartile range (IQR), 123.6-387.3 ng/mL] vs 97.6 ng/mL [IQR, 74.4-126.6 ng/mL]; P < .0001) and decreased elafin levels (32.0 ng/mL [IQR, 15.3-59.1 ng/mL] vs 45.5 ng/mL [IQR, 28.1-92.8 ng/mL]; P < .0001) independent of PAH subtype, illness duration, and therapies. Higher NE levels were associated with worse symptom severity, shorter 6-min walk distance, higher N-terminal pro-type brain natriuretic peptide levels, greater right ventricular dysfunction, worse hemodynamics, increased circulating neutrophil levels, elevated cytokine levels, and lower blood BMPR2 expression. In Stanford patients, NE levels of > 168.5 ng/mL portended increased mortality risk after adjustment for known clinical predictors (hazard ratio [HR], 2.52; CI, 1.36-4.65, P = .003) or prognostic cytokines (HR, 2.63; CI, 1.42-4.87; P = .001), and the NE level added incremental value to established PAH risk scores. Similar prognostic thresholds were identified in validation cohorts. Longitudinal NE changes tracked with clinical trends and outcomes. PAH PAECs exhibited increased apoptosis and attenuated angiogenesis when exposed to NE at the level observed in patients' blood. Elafin rescued PAEC homeostasis, yet the required dose exceeded levels found in patients.InterpretationBlood levels of NE are increased while elafin levels are deficient across PAH subtypes. Higher NE levels are associated with worse clinical disease severity and outcomes, and this target-specific biomarker could facilitate therapeutic development of elafin.

Project description:BACKGROUND:Growth Arrest and DNA Damage 45? (GADD45?) is a member of the DNA damage-inducible gene family which responds to environmental stresses. Apoptosis is a critical mode of renal tubular cell death in nephrotoxin-induced acute kidney injury. In this study, we investigated the role of GADD45? in renal tubular cell apoptosis induced by nephrotoxic drugs. METHODS:Primary human renal tubular epithelial (HRE) cells were used in this study. To derive stable cell lines in which GADD45? expression was silenced, HRE cells were transduced with a plasmid encoding GADD45?-specific shRNA. The recombinant adenovirus containing the GADD45? gene was synthesized to overexpress GADD45? protein. Cell death was induced by cisplatin and cyclosporine A (CsA). To prevent apoptotic cell death, pan-caspase inhibitor ZVAD-FMK was used. To prevent non-apoptotic cell death, necrostatin-1 and ferrostatin-1 were used. The degree of apoptosis and necrosis of cultured cells were evaluated by flow cytometry. RESULTS:Expression of the GADD45? gene was significantly upregulated in response to treatment with CsA and cisplatin. Apoptosis and necrosis induced by these drugs were significantly reduced by silencing of GADD45?, and significantly augmented by the overexpression of GADD45?. The activation of caspase-3 and caspase-7 as well as caspase-9 induced by cisplatin or CsA was reduced by silencing of GADD45?, and was augmented by the overexpression of GADD45?, indicating that caspase activation is dependent on the expression of GADD45?. ZVAD-FMK significantly inhibited apoptosis induced by cisplatin or CsA, indicating a role of caspases in mediating apoptotic cell death. ZVAD-FMK was effective to prevent necrosis as well, indicating that the observed necrosis was a secondary event following apoptosis at least in part. CONCLUSIONS:To our knowledge, this is the first study to show that GADD45? is required for the caspase-dependent apoptosis of renal tubular cells induced by nephrotoxic drugs.

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The neutrophil elastase inhibitor elafin triggers rb-mediated growth arrest and caspase-dependent apoptosis in breast cancer.

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The neutrophil elastase inhibitor elafin triggers rb-mediated growth arrest and caspase-dependent apoptosis in breast cancer.

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