Modulating protein ubiquitination via proteasome inhibition represents a promising target for cancer therapy, because of the higher sensitivity of cancer cells to the cytotoxic effects of proteasome inhibition. Here we show that CEP-18770 is a novel orally-active inhibitor of the chymotrypsin-like activity of the proteasome that down-modulates the nuclear factor-kappaB (NF-kappaB) activity and the expression of several NF-kappaB downstream effectors. CEP-18770 induces apoptotic cell death in multiple myeloma (MM) cell lines and in primary purified CD138-positive explant cultures from untreated and bortezomib-treated MM patients. In vitro, CEP-18770 has a strong antiangiogenic activity and potently represses RANKL-induced osteoclastogenesis. Importantly, CEP-18770 exhibits a favorable cytotoxicity profile toward normal human epithelial cells, bone marrow progenitors, and bone marrow-derived stromal cells. Intravenous and oral administration of CEP-18770 resulted in a more sustained pharmacodynamic inhibition of proteasome activity in tumors relative to normal tissues, complete tumor regression of MM xenografts and improved overall median survival in a systemic model of human MM. Collectively, these findings provide evidence for the utility of CEP-18770 as a novel orally active proteasome inhibitor with a favorable tumor selectivity profile for the treatment of MM and other malignancies responsive to proteasome inhibition.

Metastasis is the main reason for lung cancer-related mortality, but little is known about specific determinants of successful dissemination from primary tumors and metastasis initiation. Here, we show that CD133þ cancer-initiating cells (CIC) directly isolated from patient-derived xenografts (PDX) of non-small cell lung cancer are endowed with superior ability to seed and initiate metastasis at distant organs. We additionally report that CXCR4 inhibition successfully prevents the increase of cisplatin-resistant CD133 ), which also shows the greatest in vitro invasive potential. We next prove that recovered disseminated cells from lungs of PDX-bearing miceþ /EpCAM À CICs are highly tumorigenic and metastatic. Importantly, microenvironment stimuli eliciting epithelial-to-mesenchymal transition, including signals from cancer-associated fibroblasts, are able to increase the dissemination potential of lung cancer cells through the generation of the CD133set. These findings also have correlates in patient samples where disseminating CICs are enriched in metastatic lymph nodes (20-fold, P ¼ 0.006) and their detection in primary tumors is correlated with poor clinical outcome (diseasefree survival: P ¼ 0.03; overall survival: P ¼ 0.05). Overall, these results highlight the importance of specific cellular subsets in the metastatic process, the need for in-depth characterization of disseminating tumor cells, and the potential of therapeutic strategies targeting both primary tumor and tumor-microenvironment interactions.

Bone metastases represents a common cause of morbidity in patients suffering many types of cancer: breast, lung, kidney, prostate, and multiple myeloma. Osteolytic metastases often cause severe pain, pathologic fractures, hypercalcemia, spinal cord compression, and other nerve-compression syndromes. Osteoclasts (OCs), cells deriving from granulocitic-macrophagic lineage, are responsible for osteolysis, which may be reduced by inhibiting both OCs formation and activity. By studying bone osteolytic metastases mechanism in solid tumors, we report here our findings that cancer patients with bone involvement display an increase in osteoclasts precursors, compared with both healthy controls and cancer patients without bone metastases. Peripheral blood mononuclear cells (PBMCs) from patients with osteolytic lesions show osteoclastogenesis without adding M-CSF, RANKL, or TNF-alpha. However, these factors are necessary to generate OCs from healthy donors, non-osteolytic patient PBMCs and T-cell depleted PBMCs. OCs derived from cancer patients show more resorption pits than OCs from healthy donors and express genes involved in osteoclastogenesis. Our data show that a spontaneous osteoclastogenesis occurs in patients affected by osteolytic lesions and may be supported by factors released by T lymphocytes. These factors could give a priming to osteoclast precursors and promote osteoclastogenesis. In fact, T-cell depleted PBMCs do not differentiate into OCs without adding M-CSF and RANKL. Moreover, we do not obtain a higher number of OCs by increasing RANKL doses in cultures, and OCs and T lymphocytes mRNA level are detected for TNF-alpha but not for RANKL. The addition of OPG to PBMCs cultures do not modify spontaneous osteoclastogenesis. A neutralizing anti-TNF-alpha antibody in unstimulated PBMC cultures of osteolytic cancer patients induces an inhibition of osteoclastogenesis. These data suggest that TNF-alpha may be responsible for osteoclastogenesis in these tumors.

BackgroundBone forming metastases are a common and disabling consequence of prostate cancer (CaP). The potential role of osteoclast activity in CaP bone metastases is not completely explained. In this study, we investigated ex vivo whether the osteolytic activity is present and how it is ruled in CaP patients with bone forming metastases.MethodologyForty-six patients affected by newly diagnosed CaP and healthy controls were enrolled. At diagnosis, 37 patients had a primary tumour only, while 9 had primary tumour and concomitant bone forming metastases. In all patients there was no evidence of metastasis to other non-bone sites. For all patients and controls we collected blood and urinary samples. We evaluated patients' bone homeostasis; we made peripheral blood mononuclear cell (PBMC) cultures to detect in vitro osteoclastogenesis; we dosed serum expression of molecules involved in cancer induced osteoclatogenesis, such as RANKL, OPG, TNF-alpha, DKK-1 and IL-7. By Real-Time PCR, we quantified DKK-1 and IL-7 gene expression on micro-dissected tumour and healthy tissue sections.Principal FindingsCaP bone metastatic patients showed bone metabolism disruption with increased bone resorption and formation compared to non-bone metastatic patients and healthy controls. The CaP PBMC cultures showed an enhanced osteoclastogenesis in bone metastatic patients, due to an increase of RANKL/OPG ratio. We detected increased DKK-1 serum levels and tissue gene expression in patients compared to controls. IL-7 resulted high in patients' sera, but its tissue gene expression was comparable in patients and controls.ConclusionsWe demonstrated ex vivo that osteoclastogenesis is an active mechanism in tumour nesting of bone forming metastatic cancer and that serum DKK-1 levels are increased in CaP patients, suggesting to deeply investigate its role as tumour marker.

Nicotinamide adenine dinucleotide (NAD+) is an essential co-enzyme reported to operate both intra- and extracellularly. In the extracellular space, NAD+ can elicit signals by binding purinergic P2 receptors or it can serve as the substrate for a chain of ectoenzymes. As a substrate, it is converted to adenosine (ADO) and then taken up by the cells, where it is transformed and reincorporated into the intracellular nucleotide pool. Nucleotide-nucleoside conversion is regulated by membrane-bound ectoenzymes. CD38, the main mammalian enzyme that hydrolyzes NAD+, belongs to the ectoenzymatic network generating intracellular Ca2+-active metabolites. Within this general framework, the extracellular conversion of NAD+ can vary significantly according to the tissue environment or pathological conditions. Accumulating evidence suggests that tumor cells exploit such a network for migrating and homing to protected areas and, even more importantly, for evading the immune response. We report on the experience of this lab to exploit human multiple myeloma (MM), a neoplastic expansion of plasma cells, as a model to investigate these issues. MM cells express high levels of surface CD38 and grow in an environment prevalently represented by closed niches hosted in the bone marrow (BM). An original approach of this study derives from the recent use of the clinical availability of therapeutic anti-CD38 monoclonal antibodies (mAbs) in perturbing tumor viability and enzymatic functions in conditions mimicking what happens in vivo.

Receptor activator of NFκB ligand (RANKL), RANK, and osteoprotegerin (OPG) represent the key regulators of bone metabolism both in normal and pathological conditions, including bone metastases. To our knowledge, no previous studies investigated and compared RANK expression in primary tumors and in bone metastases from the same patient. We retrospectively examined RANK expression by immunohistochemistry in 74 bone metastases tissues from solid tumors, mostly breast, colorectal, renal, lung, and prostate cancer. For 40 cases, tissue from the corresponding primary tumor was also analyzed. Sixty-six (89%) of the 74 bone metastases were RANK-positive and, among these, 40 (59.5%) showed more than 50% of positive tumor cells. The median percentage of RANK-positive cells was 60% in primary tumors and metastases, without any statistically significant difference between the two groups (P=0.194). The same percentage was obtained by considering only cases with availability of samples both from primary and metastasis. Our study shows that RANK is expressed by solid tumors, with high concordance between bone metastasis and corresponding primary tumor. These data highlight the central role of RANK/RANKL/OPG pathway as potential therapeutic target not only in bone metastasis management, but also in the adjuvant setting.

Doxorubicin is one of the leading drugs for osteosarcoma standard chemotherapy. A total of 40% to 45% of high-grade osteosarcoma patients are unresponsive, or only partially responsive, to doxorubicin (Dox), due to the overexpression of the drug efflux transporter ABCB1/P-glycoprotein (Pgp). The aim of this work is to improve Dox-based regimens in resistant osteosarcomas. We used a chemically modified mitochondria-targeted Dox (mtDox) against Pgp-overexpressing osteosarcomas with increased resistance to Dox. Unlike Dox, mtDox accumulated at significant levels intracellularly, exerted cytotoxic activity, and induced necrotic and immunogenic cell death in Dox-resistant/Pgp-overexpressing cells, fully reproducing the activities exerted by anthracyclines in drug-sensitive tumors. mtDox reduced tumor growth and cell proliferation, increased apoptosis, primed tumor cells for recognition by the host immune system, and was less cardiotoxic than Dox in preclinical models of drug-resistant osteosarcoma. The increase in Dox resistance was paralleled by a progressive upregulation of mitochondrial metabolism. By widely modulating the expression of mitochondria-related genes, mtDox decreased mitochondrial biogenesis, the import of proteins and metabolites within mitochondria, mitochondrial metabolism, and the synthesis of ATP. These events were paralleled by increased reactive oxygen species production, mitochondrial depolarization, and mitochondria-dependent apoptosis in resistant osteosarcoma cells, where Dox was completely ineffective. We propose mtDox as a new effective agent with a safer toxicity profile compared with Dox that may be effective for the treatment of Dox-resistant/Pgp-positive osteosarcoma patients, who strongly need alternative and innovative treatment strategies. Mol Cancer Ther; 15(11); 2640-52. ©2016 AACR.

Puropose. Osteoarthritis (OA) is characterized by articular cartilage degeneration and subchondral bone sclerosis. OA can benefit of non-surgical treatments with collagenase-isolated Stromal Vascular Fraction (SVF) or cultured-expanded mesenchymal stem cells (ASCs). To avoid high manipulation of the lipoaspirate needed to obtain ASCs and SVF, we investigated whether articular infusions of autologous concentrated adipose tissue is an effective treatment for knee OA patients.Methods. The knee of 20 OA patients was intra-articularly injected with autologous concentrated adipose tissue, obtained after centrifugation of lipoaspirate. Patients' articular functionality and pain were evaluated by VAS and WOMAC scores ate 3, 6, 18 months from infusion. The osteogenic and chondrogenic ability of ASCs contained in the injected adipose tissue was studied in in vitro primary osteoblast and chondrocyte cell cultures, also plated on 3D-bone scaffold. Knee articular biopsies of patients previously treated with adipose tissue were analyzed. Immunohistochemistry (IHC) and Scanning Electron Microscopy (SEM) were performed to detect cell differentiation and tissue regeneration. Results. The treatment resulted safe, and all patients reported an improvement in terms of pain reduction and increase of function. According to the osteogenic or chondrogenic stimulation, ASCs expressed alkaline phosphatase or aggrecan, respectively. The presence of a layer of newly formed tissue was visualized by IHC staining and SEM. The biopsy of previously treated-knee joints showed new tissue formation, starting from the bone side of the osteochondral lesion. Conclusions. Overall our data indicate that adipose tissue infusion stimulates tissue regeneration and might be considered a safe treatment for knee OA.