Camilla Palumbo

Frequent overexpression of multiple ErbB receptors by head and neck squamous cell carcinoma contrasts with rare antibody immunity in patients

In an effort to elucidate the role of ErbB receptors in human head and neck squamous cell carcinoma (HNSCC), expression abnormalities and subcellular localization of epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4 were investigated along with EGF and tenascin by immunohistochemistry in 38 carcinomas as compared to adjacent normal mucosa of 24 cases. Although tumour‐specific overexpression affected each ErbB receptor (EGFR 47%, ErbB2 29%, ErbB3 21%, ErbB4 26%), EGFR abnormalities were most prevalent. The latter, and overexpression of more than two ErbB receptors in the same tumour, which always included EGFR, correlated with metastatic disease. ErbB products were specifically detected on the cell membrane and in the cytoplasm. In contrast, ErbB4 was uniquely localized to the nucleus in 7 carcinomas and a tumour‐derived cell line, indicating a role for regulated intramembrane proteolysis resulting in nuclear ErbB4 translocation in HNSCC. Expression of prototype ligand EGF or low‐affinity stromal activator tenascin correlated significantly with EGFR overexpression, implying chronic EGFR activation. Simultaneous overexpression of additional ErbB receptors in most of these cases suggested recurrent involvement of receptor heterodimers. In spite of frequent ErbB receptor alterations, autologous ErbB serum antibodies were rare, with only 1 of 38 tumour patients exhibiting an ErbB2‐specific immune response. Based on upregulation of several known immunosuppressive molecules, scarcity of ErbB‐specific antibodies is consistent with attenuation of natural tumour‐specific immune responses in HNSCC. Copyright

Expression of the PDGF α-receptor 1.5 kb transcript, OCT-4, and c-KIT in human normal and malignant tissues. Implications for the early diagnosis of testicular germ cell tumours and for our understanding of regulatory mechanisms

Human testicular germ cell tumours of adolescents and adults (TGCTs), including their precursor lesion carcinoma in situ (CIS), show expression of a 1.5 kb alternative transcript of the platelet‐derived growth factor (PDGF) α‐receptor gene. The so‐called P2 promoter involved is located in intron 12 and its activity was found to be mutually exclusive with activity of the classical promoter (P1), which encodes the full‐length receptor. The presence of the 1.5 kb transcript could be a putative marker for the early molecular diagnosis of TGCTs. In order to validate the RT‐PCR approach, this study shows that not more than 100 transcripts are necessary to obtain positivity in the test used; moreover, samples from TGCTs or CIS‐containing tissues can be diluted many‐fold before resulting in false‐negative findings. This study also shows that within TGCTs, as in TGCT‐derived cell lines, expression of the 1.5 kb transcript is differentiation‐dependent and positively correlated with expression of the embryonic transcription factor OCT‐4/POU5F1. Furthermore, the results indicate that in some non‐TGCT cancers and cell lines the 1.5 kb transcript is also expressed, but without concomitant OCT‐4/POU5F1 expression. The 1.5 kb transcript is also present in early B cells and derived leukaemias (B‐ALL). In spite of similarities in chromosomal location, down‐regulation upon differentiation of TGCTs, and PDGF α‐receptor and c‐KIT (the stem cell factor receptor) both being a tyrosine kinase receptor, no correlation was found between activity of the P2 promoter of the PDGF α‐receptor gene and expression of c‐KIT. In conclusion, the 1.5 kb transcript of the PDGF α‐receptor is expressed in various cells and tissues, including particular blood cells. Although this may hamper the use of this transcript as a marker for malignancies in general, it does not appear to interfere with assays for the early detection of TGCTs. Copyright

Apigenin induces apoptosis and impairs head and neck carcinomas EGFR/ErbB2 signaling

The development of head and neck squamous cell carcinomas (HNSCCs) is a multistep process progressing from precancerous lesions to highly malignant tumors. A critical role in HNSCCs development and progression is played by EGFR family members including EGFR and ErbB2. The aim of this study was to investigate the effect of apigenin, a low molecular weight flavonoid contained in fruits and vegetables, on growth and survival and on EGFR/ErbB2 signaling in cell lines derived from HNSCCs of the tongue (CAL-27, SCC-15) or pharynx (FaDu). Using sulforhodamine B assay, FACS analysis and activated caspase-3 detection by immunofluorescence, we here demonstrate that apigenin dose-dependently inhibits survival and induces apoptosis of HNSCC cells. Further, by performing western blotting with antibodies specific for phosphorylated EGFR, ErbB2, Erk1/2 and Akt we demonstrate that apigenin reduces ligand-induced phosphorylation of EGFR and ErbB2 and impairs their downstream signaling. On the whole, our results suggest that apigenin properties might be exploited for chemoprevention and/or therapy of head and neck carcinomas.

Stem cell activation sustains hereditary hypertrophy in hamster cardiomyopathy.

Recent studies have documented the presence of stem cells within the myocardium and their role in the repair of ischaemic injury. Nevertheless, the pathogenic role of stem cells in non‐ischaemic myocardial diseases, as well as the factors potentially responsible for their activation, is still under debate. The present study demonstrates the presence of an increased number of c‐kit positive, MDR‐positive, and Sca‐1‐positive stem cells within the myocardium of hereditary δ‐SG null hamsters, a spontaneously occurring model of hypertrophic cardiomyopathy. When hamsters are 80 days old, ie at the ‘hypertrophic’ stage of the disease, but without haemodynamic overload, these cells associate with a multitude of cells co‐expressing c‐kit, cMet, GATA4, or MEF‐2, and proliferating myocytes co‐expressing myosin heavy chain, telomerase, ki67 and cyclin B. Furthermore, at the same animal age, the number of myocardial cells co‐expressing c‐kit and Flk‐1, and the number of capillary vessels, is also amplified. In order to identify factors potentially responsible for stem cell activation, the myocardial expression of HGF and cMet and HGF plasma levels were evaluated, demonstrating their increase in 80‐day‐old δ‐SG null hamsters. To demonstrate the possible ability of HGF to induce stem cell differentiation, bone‐marrow‐derived mesenchymal stem cells were challenged with HGF at the same plasma concentration observed in vivo. HGF induced cMet phosphorylation, and caused loss of stem cell features and overexpression of MEF‐2, TEF1, and MHC. Our results demonstrate that stem cell activation occurs within the cardiomyopathic myocardium, very likely to maintain an efficient cardiac architecture. In this context, elevated levels of HGF might play a role in induction of stem cell commitment to the cardiomyocyte lineage and in cardioprotection through its anti‐apoptotic action. Consistently, when cytokine levels declined to physiological concentrations, as in 150‐day‐old cardiomyopathic animals, myocardial apoptosis prevailed, prejudicing cardiac function. Copyright

Molecular Targets and Targeted Therapies for Malignant Mesothelioma

Malignant mesothelioma is a highly invasive tumor originating from the mesothelial linings of the pleura, peritoneum and pericardium. It is seldom amenable to surgical intervention and poorly responsive to radiotherapy, leaving chemotherapy as the main therapeutic option for most patients. The development of effective drug regimens against mesothelioma has proven extremely difficult and a standard first-line treatment for patients with unresectable tumors has not been established until recently. Despite the benefits obtained with this newly validated standard of care, which is based on the combination of pemetrexed and cisplatin, the prognosis for mesothelioma patients remains poor, median survival is still less than two years and more active treatments are urgently needed. This article will focus on the molecular basis providing the rationale for targeted interventions against mesothelioma and will review targeted agents under evaluation as new potential therapeutic options for mesothelioma patients. Such agents include inhibitors of growth factor receptors, ligands and intracellular effectors. The agents targeting vascular endothelial growth factor signaling are of particular interest, due to the involvement of this pathway both in tumor angiogenesis and autocrine stimulation of mesothelioma cell growth. Alternative approaches are based on inhibitors of the ubiquitin-proteasome pathway and of histone deacetylases which, notwithstanding the functional divergence of the corresponding targets, share the ability to determine a wide modulation of the cancer cell phenotype that can lead to cell cycle arrest, apoptosis and sensitization to different antineoplastic treatments. A recombinant immunotoxin targeted to the membrane antigen mesothelin is an additional agent whose activity is being evaluated in mesothelioma patients.

Resveratrol and diallyl disulfide enhance curcumin-induced sarcoma cell apoptosis.

Malignant tumors of mesenchimal origin such as rhabdomyosarcoma and osteosarcoma are highly aggressive pedriatic malignancies with a poor prognosis. Indeed, the initial response to chemotherapy is followed by chemoresistance. Diallyl disulfide (DADS), resveratrol (RES) and curcumin (CUR) are dietary chemopreventive phytochemicals which have been reported to have antineoplastic activity on rhabdomyosarcoma and osteosarcoma cells as single drugs. In this study we evaluated whether, as compared to the single compounds, the combination of DADS+RES, DADS+CUR and RES+CUR resulted in an enhancement of their antitumor potential on malignant rhabdoid (SJ-RH4, RD/18) or osteosarcoma (Saos-2) cell lines. Through FACS analysis and activated caspase-3 labeling we demonstrate that CUR induces apoptosis of rabdomyosarcoma and osteosarcoma cells and that this effect is potentiated when CUR is combined with RES or DADS. Further, we explored the effects of the compounds, alone or in combination, on signal transduction pathways involved in apoptosis and growth of cancer cells and show that in rhabdomyosarcoma cells the apoptotic effect of CUR, either alone or in combination, is independent of p53 activity. Our findings suggest that CUR and CUR-based combinations may have relevance for the treatment of p53-deficient cancers, which are often unaffected by conventional chemotherapies or radiotherapy.

Immunity to Extracellular Matrix Antigens is Associated with Ultrastructural Alterations of the Stroma and Stratified Epithelium Basement Membrane in the Skin of Hashimoto's Thyroiditis Patients:

Employing purified extracellular matrix (ECM) proteins, i.e. type I, III, IV and V collagens (CI, CHI, CIV, CV), laminin (LM) and fibronectin (FN), as antigen sources we detected autoantibodies to conformational and/or denatured ECM antigens among 34 of 50 sera obtained from Hashimotos thyroiditis (HT) patients and 6 of 51 control sera obtained from non-autoimmune thyroid disease patients and healthy donors (HT sera vs. control sera p=4×10−9). Reactivity to conformational antigens, mostly due to autoantibodies of the IgG isotype, was observed in 30/50 HT sera and in 6/51 control sera (p=3.5×10−7) and was not always concomitant with that to linear antigens, found in 23/50 HT and in 6/51 control sera (p=1.6×10−4). Ultrastructural analysis of skin biopsies obtained from 18 HT patients without symptomatic cutaneous diseases revealed defects of the stratified squamous epithelium basement membrane in 11/18, alterations of the stroma in 13/18 and both basement membrane and stromal defects in 9/18. Interestingly, 13/13 (p=0.012) and 9/11 (p=0.012) patients with stromal and basement membrane defects respectively, exhibited serum antibodies to at least one ECM antigen involved in the organization of the altered tissue compartment. Lastly, 10/18 skin biopsies presented immunoglobulin (Ig) and/or complement (C3) deposits along the cutaneous basement membrane zone (BMZ) or in the papillary dermis and 9/10 sera from the same patients simultaneously showed antibodies to at least one ECM antigen involved in the organization of these two skin compartments. Besides, 8/11 HT patients with basement membrane defects exhibited Ig or C3 deposits along the BMZ. Our findings suggest that autoantibodies to ECM molecules might contribute to the development of asymptomatic extra-thyroid skin diseases in HT patients.

Peripheral nerve extracellular matrix remodeling in Charcot-Marie-Tooth type I disease.

Abstract. Charcot-Marie-Tooth type 1 disease (CMT1) is a group of inherited demyelinating neuropathies caused by mutations in genes expressed by myelinating Schwann cells. Rather than demyelination per se, alterations of Schwann cell-axon interactions have been suggested as the main cause of motor-sensory impairment in CMT1 patients. In an attempt to identify molecules that may be involved in such altered interactions, the extracellular matrix (ECM) remodeling occurring in CMT1 sural nerves was studied. For comparison, both normal sural nerves and sural nerves affected by neuropathies of different origin were used. The study was performed by immunohistochemical analysis using antibodies against collagen types I, III, IV, V, and VI and the glycoproteins fibronectin, laminin, vitronectin and tenascin. Up-regulation of collagens, fibronectin and laminin was commonly found in nerve biopsy specimens from patients affected by CMT1 and control diseases, but higher levels of overexpression were usually observed in CMT1 cases. On the other hand, vitronectin and tenascin appeared preferentially induced in CMT1 compared to other pathologies investigated here. Vitronectin, whose expression in normal nerves was limited to perineurial layers and to the walls of epineurial and endoneurial vessels, became strongly and diffusely expressed in the endoneurium in most CMT1 biopsy specimens. The expression of tenascin, confined to the perineurium, to vessel walls and to the nodes of Ranvier in normal nerves, was displaced and extended along the internodes of several nerve fibers in the majority of CMT1 nerves. Thus, compared with our pathological controls CMT1 seemed to determine the most extensive remodeling of peripheral nerve ECM.

Group B Streptococcus (GBS) disrupts by calpain activation the actin and microtubule cytoskeleton of macrophages

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS‐III‐COH31 in a time‐ and infection ratio‐dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS β‐haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS‐III‐COH31 in conditions that suppress β‐haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (β‐haemolysin inhibitor). Calpains, particularly m‐calpain, are responsible for GBS‐III‐COH31‐induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m‐calpain small‐interfering‐RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ‐calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS‐III‐COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS‐III‐NEM316 and GBS‐V‐10/84 through β‐haemolysin‐mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.

The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex.

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is Kd=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (Kd≥2.6 μM) or is inactivated by oxidation (Kd=1.7 μM). This indicates that GSTP1-1’s TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex’s binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1’s association with TRAF2 at the cellular level. The TRAF2–GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex’s drastic decline observed in cells progressing from the G0/G1 to the S–G2–M phases. Moreover, GSH’s intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2–GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.