Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo.

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

Tissue transglutaminase links TGF-ß, epithelial to mesenchymal transition and a stem cell phenotype in ovarian cancer.

Tissue transglutaminase (TG2), an enzyme involved in cell proliferation, differentiation and apoptosis is overexpressed in ovarian carcinomas, where it modulates epithelial-to-mesenchymal transition (EMT) and promotes metastasis. Its regulation in ovarian cancer (OC) remains unexplored. Here, we show that transforming growth factor (TGF)-ß, a cytokine involved in tumor dissemination is abundantly secreted in the OC microenvironment and induces TG2 expression and enzymatic activity. This is mediated at transcriptional level by SMADs and by TGF-ß-activated kinase 1-mediated activation of the nuclear factor-κB complex. TGF-ß-stimulated OC cells aggregate as spheroids, which enable peritoneal dissemination. We show that TGF-ß-induced TG2 regulates EMT, formation of spheroids and OC metastasis. TG2 knock-down in OC cells decreases the number of cells harboring a cancer stem cell phenotype (CD44+/CD117+). Furthermore, CD44+/CD117+ cells isolated from human ovarian tumors express high levels of TG2. In summary, TGF-ß-induced TG2 enhances ovarian tumor metastasis by inducing EMT and a cancer stem cell phenotype.

Tumor-bone cellular interactions in skeletal metastases.

Human tumor cells inoculated into the arterial circulation of immunocompromised mice can reliably cause bone metastases, reproducing many of the clinical features seen in patients. Animal models permit the identification of tumor-produced factors, which act on bone cells, and of bone-derived factors. Local interactions stimulated by these factors drive a vicious cycle between tumor and bone that perpetuates skeletal metastases. Bone metastases can be osteolytic, osteoblastic, or mixed. Parathyroid hormone-related protein, PTHrP, is a common osteolytic factor, while vascular endothelial growth factor and interleukins 8 and 11 also contribute. Osteoblastic metastases can be caused by tumor-secreted endothelin-1, ET-1. Other potential osteoblastic factors include bone morphogenetic proteins, platelet-derived growth factor, connective tissue growth factor, stanniocalcin, N-terminal fragments of PTHrP, and adrenomedullin. Osteoblasts are the main regulators of osteoclasts, and stimulation of osteoblast proliferation can increase osteoclast formation and activity. Thus, combined expression of osteoblastic and osteolytic factors can lead to mixed metastases or to increased osteolysis. Prostate-specific antigen is a protease, which can cleave PTHrP and thus change the balance of osteolytic versus osteoblastic responses to metastatic tumor cells. Bone itself stimulates tumor by releasing insulin-like growth factors and transforming growth factor-beta. Secreted factors transmit the interactions between tumor and bone. They provide novel targets for therapeutic interactions to break the vicious cycle of bone metastases. Clinically approved bisphosphonate anti-resorptive drugs reduce the release of active factors stored in bone, and PTHrP-neutralizing antibody, inhibitors of the RANK ligand pathway, and ET-1 receptor antagonist are in clinical trials. These adjuvant therapies act on bone cells, rather than the tumor cells. Recent gene array experiments identify additional factors, which may in the future prove to be clinically important targets.

Fluorescent bone viewed through toenails of living animals: a method to observe bone regrowth.

We have developed a new method to observe bone and to document growth in living animals. The technique involves injecting calcein, a fluorescent calcium deposition marker, waiting approximately 4 hr for it to clear the vascular system, and observing bone directly through the toenails of lightly anesthetized living animals. Bone regrowth can be monitored in situ by amputating the digit through the nail plate, waiting the desired number of days, and injecting a second fluorescent label, alizarin red. Bone that has regrown since the amputation appears as a red area distal to the green calcein label on toes of lightly anesthetized animals when viewed under FITC fluorescence. This method has been used to demonstrate blocked bone synthesis and to quantitate significant differences in bone growth in control and experimental toes of individual animals. Advantages of this method include its simplicity, the use of fewer animals to collect sequential data, and increased reliability of repeated microscopic measurements using the same animal.

Denervation retards but does not prevent toetip regeneration.

Toetips of mammals regrow after amputation by a process similar, but not identical, to that which occurs during regeneration of a newt limb. Nerve is needed as a mitotic stimulant for newt limb regeneration but the requirement for nerve during rodent digit-tip regeneration is not known. Nerve dependence in rats was tested by severing the sciatic nerve in one hindlimb, amputating digit-tips from the central digits of both hind feet, and comparing the amount of regrowth in innervated and denervated digits. Denervation delayed soft-tissue wound healing. However, denervation did not significantly affect bone regrowth when animals were examined at one month. Because we suspected delayed bone regrowth, we used a new method that we developed to follow bone growth at several time points in each animal. Termed visible bone fluorescence through nail, this technique used serial injections of fluorescent calcium-deposition markers and observation through the toenails to observe bone growth in living animals. Using this method it was possible to detect retarded bone regrowth in denervated digits. Thus, although denervation of rodent tips delayed both soft tissue healing and bone regrowth, it did not prevent ultimate restitution of the amputated part. This suggests that neurotrophic stimulation in the mammalian digit-tip is not identical to that documented during newt limb regeneration, and that growth stimulation may be provided by tissues other than nerve.

Bone growth is induced by nail transplantation in amputated proximal phalanges.

Mammals are able to regrow the tips of amputated fingers and toes. However, regrowth is limited to regions covered by, and is dependent upon, the presence of the nail organ. If the nail organ is responsible for bone growth in digit-tips, we reasoned that transplanted nail organ might also be able to induce outgrowth from other levels of the digit. Partial nail organ has been transplanted to amputated proximal phalanges of young rats. To date, six transplants have successfully produced outgrowth of nail. New bone growth, not seen in control amputated digits, was documented by x-ray and by alizarin red and calcein injections to be directed toward implanted nail organ. These results support an inductive role for nail organ epithelium in bone growth after amputation and provide encouragement for attempts to enhance a positive outcome after appendage amputation.

Phase contrast microscopic examination of urinary erythrocytes to localise source of bleeding: an overlooked technique?

AIMS: To localise the source of bleeding in the urinary tract in patients presenting with haematuria. METHODS: Urine samples were obtained from 109 patients with symptoms referable to the urinary tract. The sample was examined for the presence of red blood cells by phase contrast microscopy (PCM) and the proportion of dysmorphic and isomorphic red blood cells was determined. If more than 20% of the red blood cells were dysmorphic a glomerular origin for the site of bleeding was suspected; if less than 20% of the red blood cells were isomorphic a non-glomerular origin was suspected. Phase contrast microscopy and clinical findings were correlated. RESULTS: The correct bleeding site was shown in 27 of 30 (90%) patients with glomerulopathy and in all 17 patients with bleeding from the lower urinary tract, indicating that this method of analysis has a sensitivity of 90% and specificity of 100% for detecting the glomerular source of bleeding. CONCLUSIONS: The examination of urine for dysmorphic and isomorphic red blood cells by phase contrast microscopy is strongly recommended in routine clinical practice for the detection of glomerular and non-glomerular lesions. This technique may avoid unnecessary investigations for the diagnosis of the site of bleeding in patients with haematuria.