The activation of sympathetic nerves by psychosocial stress creates a favorable environment in bone for the establishment of cancer cells in a mouse model of breast cancer.

The severe defects in growth plate development caused by chondrocyte extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) gain or loss-of-function suggest that tight spatial and temporal regulation of mitogen-activated protein kinase signaling is necessary to achieve harmonious growth plate elongation and structure. We provide here evidence that neurofibromin, via its Ras guanosine triphosphatase -activating activity, controls ERK1/2-dependent fibroblast growth factor receptor (FGFR) signaling in chondrocytes. We show first that neurofibromin is expressed in FGFR-positive prehypertrophic and hypertrophic chondrocytes during growth plate endochondral ossification. Using mice lacking neurofibromin 1 (Nf1) in type II collagenexpressing cells, (Nf1 col2 2/2 mutant mice), we then show that lack of neurofibromin in post-mitotic chondrocytes triggers a number of phenotypes reminiscent of the ones observed in mice characterized by FGFR gain-offunction mutations. Those include dwarfism, constitutive ERK1/2 activation, strongly reduced Ihh expression and decreased chondrocyte proliferation and maturation, increased chondrocytic expression of Rankl, matrix metalloproteinase 9 (Mmp9) and Mmp13 and enhanced growth plate osteoclastogenesis, as well as increased sensitivity to caspase-9 mediated apoptosis. Using wildtype (WT) and Nf1 2/2 chondrocyte cultures in vitro, we show that FGF2 pulse-stimulation triggers rapid ERK1/2 phosphorylation in both genotypes, but that return to the basal level is delayed in Nf1 2/2 chondrocytes. Importantly, in vivo ERK1/2 inhibition by daily injection of a recombinant form of C-type natriuretic peptide to post-natal pups for 18 days was able to correct the short stature of Nf1 col2 2/2 mice. Together, these results underscore the requirement of neurofibromin and ERK1/2 for normal endochondral bone formation and support the notion that neurofibromin, by restraining RAS-ERK1/2 signaling, is a negative regulator of FGFR signaling in differentiating chondrocytes.

Purpose Bortezomib, a first generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response which ultimately leads to dysregulation of intracellular Ca2+ and apoptotic cell death. This study investigated the role of the Ca2+-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo. Methods Bortezomib-mediated cell death was examined using assays for apoptosis (Annexin V staining), total cell death (trypan blue exclusion) and growth inhibition (MTT). The effects of calpain on Bortezomib-induced cytotoxicity were investigated using siRNA knockdown or pharmaceutical inhibitors. Enzyme activity assays and immunofluorescence analysis were used to identify mechanistic effects. Results Inhibition of the Ca2+ dependent cysteine protease calpain, either by pharmacologic or genetic means, enhances or accelerates bortezomib-induced myeloma cell death. The increase in cell death is not associated with an increase in caspase activity, nor is there evidence of greater inhibition of proteasome activity, suggesting an alternate, calpain-regulated mechanism of brtezomib-induced cell death. Bortezomib initiates an autophagic response in myeloma cells associated with cell survival. Inhibition of calpain subverts the cytoprotective function of autophagy leading to increased bortezomib-mediated cell death. Combination therapy with bortezomib and the calpain-blocking HIV protease inhibitor, nelfinavir, reversed bortezomib resistance and induced near-complete tumor regressions in a SCID mouse xenograft model of myeloma.

Aberrant fibroblast growth factor receptor 3 (FGFR3) signaling disrupts chondrocyte proliferation and growth plate size and architecture, leading to various chondrodysplasias or bone overgrowth. These observations suggest that the duration, intensity and cellular context of FGFR signaling during growth plate chondrocyte maturation require tight, regulated control for proper bone elongation. However, the machinery fine-tuning FGFR signaling in chondrocytes is incompletely defined. We report here that neurofibromin, a Ras-GAP encoded by Nf1, has an overlapping expression pattern with FGFR1 and FGFR3 in prehypertrophic chondrocytes, and with FGFR1 in hypertrophic chondrocytes during endochondral ossification. Based on previous evidence that neurofibromin inhibits Ras-ERK signaling in chondrocytes and phenotypic analogies between mice with constitutive FGFR1 activation and Nf1 deficiency in Col2a1-positive chondrocytes, we asked whether neurofibromin is required to control FGFR1-Ras-ERK signaling in maturing chondrocytes in vivo. Genetic Nf1 ablation in Fgfr1-deficient chondrocytes reactivated Ras-ERK1/2 signaling in hypertrophic chondrocytes and reversed the expansion of the hypertrophic zone observed in mice lacking Fgfr1 in Col2a1-positive chondrocytes. Histomorphometric and gene expression analyses suggested that neurofibromin, by inhibiting Rankl expression, attenuates pro-osteoclastogenic FGFR1 signaling in hypertrophic chondrocytes. We also provide evidence suggesting that neurofibromin in prehypertrophic chondrocytes, downstream of FGFRs and via an indirect mechanism, is required for normal extension and organization of proliferative columns. Collectively, this study indicates that FGFR signaling provides an important input into the Ras-Raf-MEK-ERK1/2 signaling axis in chondrocytes, and that this input is differentially regulated during chondrocyte maturation by a complex intracellular machinery, of which neurofibromin is a critical component.

Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity. We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification. This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization in xenograft models.

The proteasome is a multi-subunit enzyme complex that plays a central role in the regulation of proteins controlling many important cellular functions including cell cycle progression, differentiation, gene transcription and apoptosis. Bortezomib is a potent proteasome inhibitor that has recently been approved for use in refractory multiple myeloma. Our previous work demonstrated that bortezomib induces an endoplasmic reticulum (ER) stress response ultimately leading to Ca2+-dependent apoptotic cell death. Intracellular Ca2+ has also been implicated in the cellular stress response known as autophagy. The biochemical events linking the cellular stress response with the induction of autophagy, and the relationship between autophagy and apoptosis are not well understood. In this study, we investigate the role of the Ca2+ dependent serine protease, calpain, as a mediator of the conversion from autophagic cell survival to accelerated cell death in the ER stress response. Using live cell imaging we demonstrate that bortezomib rapidly initiates autophagy in myeloma cells. Microtubule associated protein 1 light chain 3 beta (LC3) is an autophagic marker that is converted from the inactive form (LC3I) to the autophagosomal membrane-associated (LC3I) form. Simultaneous analysis of the LC3II/I ratio and the polyubiquitin-binding protein, p62/SQSTM1, demonstrates an early elevation in autophagosome formation with an increase in autophagic digestion. Inhibition of the Ca2+-dependent serine protease, calpain, dramatically increases the accumulation of non-functional autophagosomes illustrated by a rise in both the LC3II/I ratio and p62 levels. This effect is accompanied by a significant enhancement or acceleration of cell death. Similarly, elimination of the small catalytic subunit, CAPNS1 using siRNA significantly increased bortezomib-mediated cell death. Our data suggest that inhibition of calpain prevents autophagolysosome maturation, and subverts the survival response thereby enhancing cell death. These data suggest a new therapeutic strategy to enhance the activity of proteasome inhibitors and overcome drug resistance in refractory myeloma and other B cell malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2643. doi:10.1158/1538-7445.AM2011-2643