c-Src is a proto-oncogene, belonging to the nonreceptor protein kinases family, which plays a prominent role in carcinogenesis. In this study, we tested the hypothesis that c-Src could promote breast cancer metastasis acting on several cell types and that pharmacological disruption of its kinase activity could be beneficial for the treatment of metastases. Female BALB/cnu/nu mice were subjected to intracardiac injection of the human breast cancer cells MDA-MB-231 (MDA-231), which induced prominent bone and visceral metastases. of a c-Src kinase-dead dominant-negative construct (MDA-231-Src DN ) resulted in reduced morbidity, lethality, and incidence of metastases similar to the mice treated with the inhibitor. An analogous beneficial effect of c-Src inhibition was observed in subcutaneous and intratibial implanted tumors. In vitro, c-Src suppression reduced MDA-231 cell aggressiveness. It also impaired osteoclast bone resorption both directly and by reducing expression by osteoblasts of the osteoclastogenic cytokines interleukin-1␤ and interleukin-6, whereas parathyroid hormone-related peptide was not implicated. c-Src was also modestly but consistently involved in the enhancement of endothelial cell proliferation in vitro and angiogenesis in vivo. In conclusion, we propose that c-Src disruption affects the metastatic process and thus is a therapeutic target for the treatment of breast cancer.Breast cancer is a relatively common tumor, with an estimated incidence of 1.2 million new cases diagnosed worldwide every year (Henderson et al., 1993). The outcome of patients mainly depends on the development of distant metastases (Greenberg et al., 1996). Bone is the principal metastatic site in patients with mammary carcinomas (James et al., 2003), of whom approximately 20% survives for more than 5 years, whereas those with minor metastases in the bone can survive up to 10 years or more. In contrast, visceral metastases, although less common, are more likely to be fatal with a higher risk of early death.Consistent evidence suggests the involvement of the protooncogene c-Src in the development and progression of many human cancers, including breast carcinomas (Otthenoff-Kalff et This work was supported by the E.C. Grant METABRE (LSHM-CT-2003-503049) and by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC).Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

Mechanical unloading causes detrimental effects on the skeleton, but the underlying mechanisms are still unclear. We investigated the effect of microgravity on osteoblast ability to regulate osteoclastogenesis. Mouse osteoblast primary cultures were grown for 24 h at unit gravity or under simulated microgravity, using the NASA-developed Rotating Wall Vessel bioreactor. Conditioned media (CM) from osteoblasts subjected to microgravity increased osteoclastogenesis and bone resorption in mouse bone marrow cultures. In these osteoblasts, the RANKL/OPG ratio was higher relative to 1g. Consistently, treatment with high concentrations of OPG-inhibited osteoclastogenesis and bone resorption in the presence of CM arising from osteoblasts cultured under microgravity. Microgravity failed to affect osteoblast differentiation and function in the time frame of the experiment, as we found no effect on alkaline phosphatase mRNA and activity, nor on Runx2, osteocalcin, osteopontin, and collagen1A2 mRNA expression. In contrast, microgravity induced a time dependent increase of ERK-1/2 phosphorylation, while phospho-p38 and phospho-JNK remained unchanged. Apoptosis, revealed by bis-benzimide staining, was similar among the various gravity conditions, while it was increased under microgravity after treatment with the MEK-1/2 inhibitor, PD98059, suggesting a protection role by ERK-1/2 against cell death. In conclusion, microgravity is capable to indirectly stimulate osteoclast formation and activity by regulating osteoblast secretion of crucial regulatory factors such as RANKL and OPG. We hypothesize that this mechanism could contribute to bone loss in individuals subjected to weightlessness and other unloading conditions.

The PRELP heparin sulfate–binding protein translocates to the nucleus, where it impairs NF-κB transcriptional activity, which in turn regulates bone homeostasis.

Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid β-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.

It has been postulated that glutathione S-transferases (GST; EC 2.5.1.18) may play a role in protecting against oxidative stress. In previous studies, we have purified and characterised from Bufo bufo embryos a GST isoenzyme (BbGSTP1-1), which falls at very low level in the adult liver, where a novel isoform (BbGSTP2-2), starts to be highly expressed. During transition to adult life, B. bufo leaves the aquatic environment to live predominantly in the terrestrial environment, characterised by higher oxygen concentration. It has been found that BbGSTP2-2 is more efficient in scavenging from organic hydroperoxides. Therefore, the appearance of BbGSTP2-2 may respond to the necessity of providing the adult toad with a more suitable protection against oxygen toxic by-products. In this work, we performed experiments aimed at verifying if oxidative stress (hyperoxic and H(2)O(2) treatments) could act as a modulator of BbGSTP2-2 expression. Results show that: (a) BbGSTP2 mRNA starts to be expressed in the late embryonic period, while protein appears during metamorphosis; (b) oxidative stress induces anticipation of BbGSTP2 gene expression at both transcriptional and translational levels. These findings seem to indicate that the appearance of BbGSTP2-2 is aimed at endowing the adult toad with more efficient antioxidant defence in the terrestrial atmosphere.

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Purpose: The tyrosine kinase inhibitor (TKI) sunitinib is a multi-targeted agent approved across multiple cancer indications. Nevertheless, since approval, data has emerged to describe a worrisome side effect profile including hypertension, hand-foot syndrome, fatigue, diarrhea, mucositis, proteinuria, and (rarely) congestive heart failure. It has been hypothesized that the observed multi-parameter toxicity profile is related to "on-target" kinase inhibition in "off-target" tissues. Experimental Design: To interrogate off-target effects in pre-clinical studies, a reverse phase protein array (RPPA) approach is employed. Mice are treated with sunitinib (40 mg kg −1 ) for 4 weeks, following which critical organs are removed. The Zeptosens RPPA platform is employed for protein expression analysis. Results: Differentially expressed proteins associated with damage and/or stress are found in the majority of organs from treated animals. Proteins differentially expressed in the heart are associated with myocardial hypertrophy, ischaemia/reperfusion, and hypoxia. However, hypertrophy is not evidenced on histology. Mild proteinuria is observed; however, no changes in renal glomerular structure are visible via electron microscopy. In skin, proteins associated with cutaneous inflammation, keratinocyte hyper-proliferation, and increased inflammatory response are differentially expressed. Conclusions and Clinical Relevance: It is posited that pre-clinical implementation of a combined histopathological/RPPA approach provides a sensitive method to mechanistically elucidate the early manifestation of TKI on-target/organ offtarget toxicities.

Background: Sunitinib is a multi-targeted agent approved for treatment of a number of cancers. However, since approval, data has continually emerged relating to toxicity profiles including hypertension, hand-foot syndrome and fatigue. Underlying mechanisms are unresolved. It has been hypothesised that the multi-parameter toxicity profile is related to ‘on-target’ kinase inhibition in ‘off-target’ tissues. Methods: To interrogate off-target effects, the Zeptosens Reverse Phase protein Array (RPA) platform was used to assess tissues obtained from mice treated with sunitinib (40 mg/kg) for 4 weeks. Additional tissue was collected for histological analyses and pathophysiologic changes assessed. Results: RPA data analyses on all organs collected (heart, kidney, liver, brain, intestine and skin) revealed the presence of differentially expressed proteins associated with damage and/or stress. Of note, Cyclin D1, MEK and phosphorylated-p21(CIP/WAF1) expression increased by 1.8-fold (p < 0.05), 2.2-fold (p < 0.1) and 1.7-fold (p < 0.1) respectively in the kidneys of sunitinib treated mice. These proteins are associated with kidney damage after chemotherapy, and induction of genes associated with renal immune response, inflammation and apoptosis. Mild proteinuria was observed in sunitinib treated animals, however no gross change in renal glomerular ultrastructure was visible via electron microscopy. Phosphorylated-FGFR1, cleaved Caspase-7, and phosphorylated-cMYC expression decreased by 0.45-fold (p < 0.05), 0.4-fold (p < 0.1) and 0.8-fold (p < 0.1) respectively in the skin of sunitinib treated mice. Down-regulation of these proteins has been associated with a perturbation of cutaneous wound healing (p-FGFR1/cleaved Caspase-7). Mice treated with high dose sunitinib (80 mg/kg) showed overt signs of skin toxicity. Histopathologic studies of the thyroid gland revealed decreased thyroid epithelial cell height (p < 0.05). Nevertheless, serum levels of triiodothyronine and thyroxine remained unchanged. An orthogonal validation study (Western blotting) is ongoing based on gene ontology, pathway analysis and observed fold changes in critical signalling pathway proteins involved in cellular stress. Conclusions: Implementation of a combined histopathologic/ RPA approach may provide a sensitive method to mechanistically elucidate the off-target toxicity sequelae associated with TKIs approved in the oncology setting. Work was supported by an EU funded Industry Academia Pathways and Partnerships Marie Curie Award (AngioTox) Grant Number 251528. RPAs were performed at Bayer Technology Services, Leverkusen, Germany. Citation Format: Alice C. O’Farrell, Adam Lafferty, Ian Miller, Rhys Evans, Maurice Cary, David Murray, Marina Alamanou, Girish Mallya Udupi, Liam Shiels, William M. Gallagher, Annette T. Byrne. Proteomic analysis of off-target toxicities following treatment with the tyrosine kinase inhibitor sunitinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3010.