Cancer-associated muscle weakness is poorly understood and there is no effective treatment. Here, we find that seven different mouse models of human osteolytic bone metastases, representing breast, lung and prostate cancers, as well as multiple myeloma exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that TGF-β, released from the bone surface as a result of metastasis-induced bone destruction upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor/calcium (Ca2+) release channel (RyR1). The oxidized RyR1 channels leaked Ca2+, resulting in lower intracellular signaling required for proper muscle contraction. We found that inhibiting RyR1 leak, TGF-β signaling, TGF-β release from bone or Nox4 all improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast cancer- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, higher levels of Nox4 protein and Nox4 binding to RyR1, and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a non-malignant metabolic bone disorder associated with increased TGF-β activity. Thus, metastasis-induced TGF-β release from bone contributes to muscle weakness by decreasing Ca2+-induced muscle force production.

Cachexia affects the majority of cancer patients, with currently no effective treatments. Cachexia is defined by increased fatigue and loss of muscle function resulting from muscle and fat depletion. Previous studies suggest that chemotherapy may contribute to cachexia, although the causes responsible for this association are not clear. The purpose of this study was to investigate the mechanism(s) associated with chemotherapy-related effects on body composition and muscle function. Normal mice were administered chemotherapy regimens used for the treatment of colorectal cancer, such as Folfox (5-FU, leucovorin, oxaliplatin) or Folfiri (5-FU, leucovorin, irinotecan) for 5 weeks. The animals that received chemotherapy exhibited concurrent loss of muscle mass and muscle weakness. Consistently with previous findings, muscle wasting was associated with up-regulation of ERK1/2 and p38 MAPKs. No changes in ubiquitin-dependent proteolysis or in the expression of TGFβ-family members were detected. Further, marked decreases in mitochondrial content, associated with abnormalities at the sarcomeric level and with increase in the number of glycolytic fibers were observed in the muscle of mice receiving chemotherapy. Finally, ACVR2B/Fc or PD98059 prevented Folfiri-associated ERK1/2 activation and myofiber atrophy in C2C12 cultures. Our findings demonstrate that chemotherapy promotes MAPK-dependent muscle atrophy as well as mitochondrial depletion and alterations of the sarcomeric units. Therefore, these findings suggest that chemotherapy potentially plays a causative role in the occurrence of muscle loss and weakness. Moreover, the present observations provide a strong rationale for testing ACVR2B/Fc or MEK1 inhibitors in combination with anticancer drugs as novel strategies aimed at preventing chemotherapy-associated muscle atrophy.

Enveloped viruses form at cellular membranes of infected cells by a budding process. Soluble viral components, along with integral membrane glycoprotein spikes, assemble at highly concentrated budding sites, followed by envelopment of the viral core and fission of the membrane to create progeny virions. Much progress has been made in identifying both viral and cellular components that drive this assembly process. For example, alphavirus budding has been found to require both soluble nucleocapsid cores and envelope glycoproteins (9,25,44,45), and a specific interaction between the viral capsid protein and the cytoplasmic tail of the viral E2 glycoprotein is critical for assembly (54). In contrast, the assembly and budding of retroviruses does not require participation of envelope components, and expression of soluble Gag polyprotein in the absence of any other viral component results in efficient budding of virus-like particles (VLPs) that resemble immature virions (46). Several elements within the Gag protein sequence that are important for budding have been identified, including M domains which mediate membrane targeting (1, 31, 32), I domains which direct Gag-Gag interactions (3, 11), and L domains which recruit cellular machinery necessary for membrane fission and virus release (1, 12, 14, 34-36, 41, 43, 48, 49).The parameters that influence the budding of negativestrand RNA viruses are not as well defined. A key role is played by soluble matrix (M) proteins that bind to the inner surfaces of plasma membranes and form an electron-dense layer underlying the virion envelope. Recombinant viruses that lack M proteins have been constructed in the cases of both rabies (27) and measles (4), and in both cases budding was drastically impaired. Furthermore, VLP release has been observed from cells transfected with plasmids encoding M proteins derived from vesicular stomatitis virus (VSV) (16,21,23), Ebola virus (15,47), influenza A virus (13,22), and human parainfluenza virus type 1 (hPIV-1) (5). VLP budding has been examined quantitatively for both VSV (21) and Ebola virus (47), and in both cases budding was remarkably efficient, with Ͼ20% of the M protein released from transfected cells into the culture medium. VLP budding directed by the M proteins of influenza A virus and hPIV-1 has at this point been examined only qualitatively (5,13,22). In all of these cases, budding of particles was observed upon expression of M protein in the absence of any other viral components. However, studies with recombinant viruses, including recombinant rabies virus (26), VSV (40), influenza A virus (20), and the paramyxoviruses Sendai virus (10) and simian virus 5 (SV5) (39) suggest that M proteins may not be sufficient to direct normal budding of a virus, since truncations or sequence alterations to glycoprotein cytoplasmic tails resulted in poor budding, despite the presence of unmodified matrix protein. SV5, like other paramyxoviruses, consists of a core of genomic RNA encapsidated by nucleocapsid (NP) protein that

Bone metastases occur in ~70% of metastatic breast cancer patients often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3) which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n=42). Mechanistic investigations revealed that TGF-β signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ~12.5nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared to equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared to free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site.