Human and mouse granzyme A induce a proinflammatory cytokine response.

Granzyme A (GzmA) is considered a major proapoptotic protease. We have discovered that GzmA-induced cell death involves rapid membrane damage that depends on the synergy between micromolar concentrations of GzmA and sublytic perforin (PFN). Ironically, GzmA and GzmB, independent of their catalytic activity, both mediated this swift necrosis. Even without PFN, lower concentrations of human GzmA stimulated monocytic cells to secrete proinflammatory cytokines (interleukin-1beta [IL-1beta], TNFalpha, and IL-6) that were blocked by a caspase-1 inhibitor. Moreover, murine GzmA and GzmA(+) cytotoxic T lymphocytes (CTLs) induce IL-1beta from primary mouse macrophages, and GzmA(-/-) mice resist lipopolysaccharide-induced toxicity. Thus, the granule secretory pathway plays an unexpected role in inflammation, with GzmA acting as an endogenous modulator.

Dual blockade of CD47 and HER2 eliminates radioresistant breast cancer cells.

Although the efficacy of cancer radiotherapy (RT) can be enhanced by targeted immunotherapy, the immunosuppressive factors induced by radiation on tumor cells remain to be identified. Here, we report that CD47-mediated anti-phagocytosis is concurrently upregulated with HER2 in radioresistant breast cancer (BC) cells and RT-treated mouse syngeneic BC. Co-expression of both receptors is more frequently detected in recurrent BC patients with poor prognosis. CD47 is upregulated preferentially in HER2-expressing cells, and blocking CD47 or HER2 reduces both receptors with diminished clonogenicity and augmented phagocytosis. CRISPR-mediated CD47 and HER2 dual knockouts not only inhibit clonogenicity but also enhance macrophage-mediated attack. Dual antibody of both receptors synergizes with RT in control of syngeneic mouse breast tumor. These results provide the evidence that aggressive behavior of radioresistant BC is caused by CD47-mediated anti-phagocytosis conjugated with HER2-prompted proliferation. Dual blockade of CD47 and HER2 is suggested to eliminate resistant cancer cells in BC radiotherapy.

Granzyme A Contributes to Inflammatory Arthritis in Mice Through Stimulation of Osteoclastogenesis.

OBJECTIVE: Granzyme A (GzmA) levels are elevated in the plasma and synovium of patients with rheumatoid arthritis (RA), suggesting involvement of this protease in the pathogenesis of the disease. GzmA contributes to sepsis by regulating the production of proinflammatory cytokines. The purpose of this study was to evaluate the contribution of GzmA to the pathogenesis of RA in vivo and to examine the possibility that GzmA acting via tumor necrosis factor (TNF) stimulates osteoclastogenesis. METHODS: Inflammatory arthritis induced by type II collagen was evaluated in wild-type, GzmA-deficient, and perforin-deficient mice. The osteoclastogenic potential of GzmA was examined in vitro using bone marrow cells and colony-forming unit-granulocyte-macrophage (CFU-GM) cells and in vivo using GzmA-deficient mice. RESULTS: Gene deletion of GzmA attenuated collagen-induced arthritis, including serum levels of proinflammatory cytokines, joint damage, and bone erosion in affected mice, suggesting that osteoclast activity is reduced in the absence of GzmA. Accordingly, GzmA-treated bone marrow cells produced multinucleated cells that fulfilled the criteria for mature osteoclasts: tartrate-resistant acid phosphatase (TRAP) activity, ß integrin expression, calcitonin receptor expression, and resorptive activity on dentin slices. GzmA appeared to act without accessory cells, and its activity was not affected by osteoprotegerin, suggesting a minor contribution of RANKL. It also induced the expression and secretion of TNF. Neutralization of TNF or stimulation of CFU-GM cells from TNF-/- mice prevented GzmA-induced osteoclastogenesis. GzmA-deficient mice had reduced osteoclastogenesis in vivo (fewer calcitonin receptor-positive multinucleated cells and fewer transcripts for cathepsin K, matrix metalloproteinase 9, and TRAP in joints) and reduced serum levels of C-terminal telopeptide of type I collagen. CONCLUSION: GzmA contributes to the joint destruction of RA partly by promoting osteoclast differentiation.

Paget's disease-a VDR coactivator disease?

Paget's disease is the most exaggerated example of bone remodeling with increased osteoclastic bone resorption followed by excessive bone formation. One of the earliest findings in our studies of Paget's disease is that pagetic osteoclast (OCL) precursors are hyper-responsive to 1,25-(OH)(2)D(3) and form OCL at concentrations of 1,25-(OH)(2)D(3) that are physiologic rather than pharmacologic. The increased responsivity to 1,25-(OH)(2)D(3) is not due to increased levels of the Vitamin D receptor (VDR) or to increased infinity of 1,25-(OH)(2)D(3) for VDR. We have recently shown using GST-VDR chimeric protein pull-down assays that TAF(II)-17, a member of the TAF(II)-D transcription complex, is increased in OCL precursors from patients with Paget's disease compared to normals. We further showed that TAF(II)-17 can enhance VDR mediated gene transcription and allow formation of the transcription complex at very low levels of 1,25-(OH)(2)D(3). In addition, coactivators of VDR including CPB300 and DRIP205 are also increased in OCL precursors from Paget's patients. These data suggest that the enhanced sensitivity of OCL precursors for 1,25-(OH)(2)D(3) in Paget's disease results from increased expression of coactivators of VDR and suggest that part of the pathophysiology underlying OCL formation in Paget's disease may result from enhanced expression of VDR coactivators.

HER2-associated radioresistance of breast cancer stem cells isolated from HER2-negative breast cancer cells.

PURPOSE: To understand the role of HER2-associated signaling network in breast cancer stem cells (BCSC) using radioresistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted therapy as a tumor eliminating strategy for recurrent HER2(-/low) breast cancers. EXPERIMENTAL DESIGN: HER2-expressing BCSCs (HER2(+)/CD44(+)/CD24(-/low)) were isolated from radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggressiveness and radioresistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer specimens. Protein expression profiling in HER2(+)/CD44(+)/CD24(-/low) versus HER2(-)/CD44(+)/CD24(-/low) BCSCs was conducted with two-dimensional difference gel electrophoresis (2-D DIGE) and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) analysis and HER2-mediated signaling network was generated by MetaCore program. RESULTS: Compared with HER2-negative BCSCs, HER2(+)/CD44(+)/CD24(-/low) cells showed elevated aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by Matrigel invasion, tumor sphere formation, and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the HER2(+)/CD44(+)/CD24(-/low) cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin treatments. Clinical breast cancer specimens revealed that cells coexpressing HER2 and CD44 were more frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC/MS-MS of HER2(+)/CD44(+)/CD24(-/low) versus HER2(-)/CD44(+)/CD24(-/low) BCSCs reported a unique HER2-associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial function, and DNA repair. A specific feature of HER2-STAT3 network was identified. CONCLUSION: This study provides the evidence that HER2-mediated prosurvival signaling network is responsible for the aggressive phenotype of BCSCs that could be targeted to control the therapy-resistant HER2(-/low) breast cancer.

A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells.

The molecular interaction of secreted granzyme B-serglycin complexes with target cells remains undefined. Targets exposed to double-labeled granzyme B-serglycin complexes show solely the uptake of granzyme B. An in vitro model demonstrates the exchange of the granzyme from serglycin to immobilized, sulfated glycosaminoglycans. Using a combination of cell binding and internalization assays, granzyme B was found to exchange to sulfated glycosaminoglycans and, depending on the cell type, to higher affinity sites. Apoptosis induced by purified granzyme B and cytotoxic T-cells was diminished in targets with reduced cell surface glycosaminoglycan content. A mechanism of delivery is proposed entailing electrostatic transfer of granzyme B from serglycin to cell surface proteins.