Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy, structure, and weakness.

Cachexia is a relevant comorbid condition of chronic diseases including cancer. Inflammation, oxidative stress, autophagy, ubiquitin-proteasome system, nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) are involved in the pathophysiology of cancer cachexia. Currently available treatment is limited and data demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF-κB, and MAPK inhibitors improve muscle mass and function loss through several molecular mechanisms. Body and muscle weights, limb muscle force, protein degradation and the ubiquitin-proteasome system, signaling pathways, oxidative stress and inflammation, autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing cachectic mice (BALB/c), with and without concomitant treatment with NF-κB (sulfasalazine), MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins, autophagy, myostatin, protein oxidation, FoxO-1, NF-κB and MAPK signaling pathways, and muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow- and fast-twitch muscle fiber size were decreased. Pharmacological inhibition of NF-κB and MAPK, but not the proteasome system, induced in cancer cachectic animals, a substantial restoration of muscle mass and force through a decrease in muscle protein oxidation and catabolism, myostatin, and autophagy, together with a greater content of myogenin, and contractile and functional proteins. Attenuation of MAPK and NF-κB signaling pathway effects on muscles is beneficial in cancer-induced cachexia.

Mitochondrial dysfunction and therapeutic approaches in respiratory and limb muscles of cancer cachectic mice.

NEW FINDINGS: What is the central question of this study? We explored whether experimental cancer-induced cachexia may alter mitochondrial respiratory chain (MRC) complexes and oxygen uptake in respiratory and peripheral muscles,and whether signalling pathways, proteasome and oxidative stress influence that process. What is the main finding and what is its importance? In cancer cachectic mice, MRC complexes and oxygen consumption were decreased in the diaphragm and gastrocnemius. Blockade of nuclear factor-κB and mitogen-activated protein kinase actions partly restored the muscle mass and force and corrected the MRC dysfunction,while concomitantly reducing tumour burden. Antioxidants improved mitochondrial oxygen consumption without eliciting effects on the loss of muscle mass and force or the tumour size,whereas bortezomib reduced tumour burden without influencing muscle mass and strength or MRC function. Abnormalities in mitochondrial content, morphology and function have been reported in several muscle-wasting conditions. We specifically explored whether experimental cancer-induced cachexia may alter mitochondrial respiratory chain (MRC) complexes and oxygen uptake in respiratory and peripheral muscles, and whether signalling pathways, proteasomes and oxidative stress may influence that process. We evaluated complex I, II and IV enzyme activities (specific activity assays) and MRC oxygen consumption (polarographic measurements) in diaphragm and gastrocnemius of cachectic mice bearing the LP07 lung tumour, with and without treatment with N-acetylcysteine, bortezomib and nuclear factor-κB (sulfasalazine) and mitogen-activated protein kinases (MAPK, U0126) inhibitors (n = 10 per group for all groups). Whole-body and muscle weights and limb muscle force were also assessed in all rodents at baseline and after 1 month. Compared with control animals, cancer cachectic mice showed a significant reduction in body weight gain, smaller sizes of the diaphragm and gastrocnemius, lower muscle strength, decreased activity of complexes I, II and IV and decreased oxygen consumption in both muscles. Blockade of nuclear factor-κB and MAPK actions restored muscle mass and force and corrected the MRC dysfunction in both muscles, while partly reducing tumour burden. Antioxidants improved mitochondrial oxygen uptake without eliciting significant effects on the loss of muscle mass and force or tumour size, whereas the proteasome inhibitor reduced tumour burden without significantly influencing muscle mass and strength or mitochondrial function. In conclusion, nuclear factor-κB and MAPK signalling pathways modulate muscle mass and performance and MRC function of respiratory and limb muscles in this model of experimental cancer cachexia, thus offering targets for therapeutic intervention.

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer.

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti-NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti-NCAM-treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM-140 knocked-down by siRNA in LP07 cells pretreated or not with anti-NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti-NCAM treatment could be mimicking homophilic trans-interactions and NCAM-140 knocked-down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression.

Opposite effects of protein kinase C beta1 (PKCbeta1) and PKCepsilon in the metastatic potential of a breast cancer murine model.

In this paper we investigated whether protein kinase C (PKC) beta1 and PKCepsilon, members of the classical and novel PKC family, respectively, induce phenotypic alterations that could be associated with tumor progression and metastatic dissemination in a murine model of breast cancer. Stable overexpression of PKCbeta1 in LM3 cells altered their ability to proliferate, adhere, and survive, and impaired their tumorigenicity and metastatic capacity. Moreover, PKCbeta1 induced the re-expression of fibronectin, an extracellular matrix glycoprotein which loss has been associated with the acquisition of a transformed phenotype in different cell models, and exerted an important inhibition on proteases production, effects that probably impact on LM3 invasiveness and dissemination. Conversely, PKCepsilon overexpression enhanced LM3 survival, anchorage-independent growth, and caused a significant increase in spontaneous lung metastasis. Our results suggest PKCbeta1 functions as an inhibitory protein for tumor growth and metastasis dissemination whereas PKCepsilon drives metastatic dissemination without affecting primary tumor growth.

Protein kinase C delta enhances proliferation and survival of murine mammary cells.

Protein kinase C (PKC) delta, a member of the novel family of PKC serine-threonine kinases, has been implicated in negative regulation of proliferation and apoptosis in a large number of cell types, including breast cancer cell lines, and postulated as a tumor suppressor gene. In this study we show that in murine NMuMG mammary cells PKCdelta promotes a mitogenic response. Overexpression of PKCdelta in NMuMG cells leads to a significant increase in [3H]-tymidine incorporation and cell proliferation, as well as enhanced extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) activation. Activation of PKCdelta with a phorbol ester leads to elevated cyclin D1 expression and an hyperphosphorylated Rb state. Surprisingly, ectopic expression of PKCdelta conferred anchorage-independent growth capacity to NMuMG cells. PKCdelta overexpressors showed enhanced resistance to apoptotic stimuli, such as serum deprivation or doxorubicin treatment, an effect that correlates with hyperactivation of the Akt survival pathway. Our results provide evidence for a role of PKCdelta as a positive modulator of proliferative and survival signals in immortalized mammary cells. The fact that PKCdelta exerts differential responses depending on the cell context not only highlights the necessity to carefully understand the signaling events controlled by this PKC in each cell type but also suggests that we should be cautious in considering this kinase a target for cancer therapy.