The Proteasome Inhibitor Bortezomib Induces Tongue, Pharynx and Salivary Gland Cancer Cells Death in Vitro and Delays Tumor Growth of Salivary Gland Cancer Cells Transplanted in Mice

Head and neck cancer (HNC) has frequently an aggressive course for the development of resistance to standard chemotherapy. Thus, the use of innovative therapeutic drugs is being assessed. Bortezomib is a proteasome inhibitor with strong in vitro and in vivo anticancer effects. In vitro antitumoral activity of Bortezomib was investigated employing human pharynx (FaDu), tongue (SCC-15, CAL-27), salivary gland (A-253) cancer cell lines and a murine cell line (SALTO-5) originated from a salivary gland adenocarcinoma arising in BALB-neuT male mice transgenic for the oncogene neu. Bortezomib in vivo effects in BALB-neuT mice transplanted with murine SALTO-5 cells were also examined. Bortezomib inhibited cells proliferation, triggered apoptosis, modulated the expression and activation of pro-survival signal transduction pathways proteins activated by ErbB receptors and inhibited proteasome activity in vitro. Furthermore, intraperitoneal administration of Bortezomib delayed tumor growth of SALTO-5 cells transplanted in BALB-neuT mice and protracted mice survival. Our ndings further support the use of Bortezomib for the treatment of HNC and reveal its ineffectiveness in counteracting the activation of deregulated specic signaling pathways in HNC cell lines when resistance to proteasome inhibition is developed. Tumors from three animals from each group of mice were used for histological examination after hematoxylin/eosin staining using 3 µm thick paran sections. Necrotic areas were measured using ImageJ software on 10 microscopic elds. Immunohistochemistry (IHC) was used to analyze the presence of caspase 3-positive cells (apoptotic cells) and the expression of ErbB2, AKT and phospho-AKT in tumors from DMSO- and Bortezomib-treated mice 109 . For IHC, antigen retrieval was performed on 3 µm thick paran sections using EDTA citrate, pH 7.8, or citrate pH 6.0 buffers for 30 minutes at 98°C. Sections were then incubated for 1 hour at room temperature (anti-cleaved caspase 3 and anti-ErbB2 antibodies) or overnight at 4°C (anti-AKT and anti-phospho-AKT) with primary antibodies. To remove non-specic binding, slides were washed using PBS/Tween 20, pH 7.6. Antibody-antigen binding was revealed by the Horseradish Peroxidase-3,3-diaminobenzidine (HRP-DAB) Detection Kit (UCS Diagnostic, Rome, Italy). The count of cleaved caspase 3-positive cells (apoptotic cells) was performed on 10 microscopic elds at 200x magnication. Semiquantitative ErbB2, AKT and phospho-AKT expression in tumors derived from DMSO- and Bortezomib-treated mice was estimated at x200 magnication in at least 10 the induction of apoptosis in several types of cancer . Our ndings showed that the treatment with Bortezomib was able to inhibit the phosphorylation of ERK1 and/or ERK2 in FaDu, SCC-15 and A-253 cells. Our results showed that the effect of Bortezomib on the modulation of the p38 activation was cell lines dependent. Bortezomib induced an increase in the phosphorylated form of JNK p54 and/or p46 in CAL-27, SCC-15, and A-253 cells but not in FaDu cells. This nding corroborated other studies in which it has been shown that Bortezomib induced apoptosis by activating JNK kinase 79 in multiple myeloma 80, 81 and in non-small cell lung cancer 82 . Our nding extends this observation to the salivary gland adenocarcinomas cell line. In addition, other studies have shown that the activation of JNK kinase is necessary for the activation of death by autophagy in HNSCC cell lines 47,58 . Accordingly, our results showed that Bortezomib induced autophagy in human HNSCC cells, but the process was then blocked, as showed by the increase of p62 39 . The same effect was observed for the rst time in the salivary gland adenocarcinoma cell line. The block of the autophagic ux by Bortezomib was reported in ovarian cancer cells, hepatocellular carcinoma cells and endometrial cancer cells 83 , breast cancer cells 84 , and B-Raf-mutated melanoma cells 85 .


Introduction
Head and neck cancer (HNC) is the seventh leading cause of cancer-related morbidity and mortality, with 890000 new cases diagnosed every year 1,2 . HNC involves several anatomical sites including pharynx, larynx, oral cavity, tongue and salivary glands 2 and often has an aggressive course with emerging resistance to conventional chemotherapy. Salivary gland carcinomas represent 6-8% of HNC, with heterogeneous morphologies and clinical outcomes 3 . The development of new targeted therapies has opened new scenarios for the treatment of solid malignancies 4,5 . Therefore, use of new therapeutic agents is being evaluated in HNC as well [6][7][8][9] . Current targeted regimen for HNC patients employs epidermal growth factor receptor (EGFR) inhibitor(s) and the monoclonal antibody cetuximab 1,10 .
However, despite promising outcomes in preclinical studies, resistance to anti-EGFR targeted therapies in clinical settings represents a limitation for prolonged use of these drugs 7,11,12 . Among different targeted therapies, inhibitors of the Ubiquitin Proteasome System (UPS), and in particular of the proteasome, are assuming particular importance 13 . They have become a new relevant class of drugs for treatment of tumors, regulation of the immune response and as anti-in ammatory agents 14,15 .
The UPS is a major intracellular proteolytic pathway 16 . The substrate, usually early-lived proteins, is conjugated to a chain of at least four ubiquitin (Ub) moieties by E1-E2-E3 enzymes and then delivered to the proteasome, a multi-catalytic assembly, for its enzymatic processing 17 . In the canonical structural con guration, the proteasome is made up by the 19S RP (i.e., Regulatory Particle) which carries out the recognition of poly-ubiquitinated substrates (poly-Ub) and couples their ATP-dependent unfolding with the translocation into the catalytic chamber of the 20S CP (i.e., Core Particle) 15,17 . Thereafter, the substrate is digested through three enzymatic activities (i.e., chymotrypsin-like, trypsin-like and caspaselike, housed by the β5, β2 and β1 subunits, respectively) 18 . Mature 20S and 19S particles can assemble into different structural con gurations, which display an emerging metabolic relevance since they are thought to deal with clearance of different subsets of natural substrates; in particular, poly-ubiquitinated proteins are fed to capped assemblies, whereas oxidized, misfolded or natively unfolded proteins are cleared by the 20S CP 17,19 . Nonetheless, the 20S/capped assemblies ratio has been uncovered to be nely tuned depending on the metabolic need of the cell and every living cell displays proteasome assemblies in the most suited con guration to cope with the maintenance of the proteostasis network (PN). PN de nes the equilibrium between protein synthesis and degradation which must be kept unaltered over the cell life cycle to guarantee cell viability and proliferation. Given the central relevance of the UPS in regulating this key network, several pharmacological strategies have been undertaken over the last decades to design drugs which speci cally target proteasome proteolytic activity. The purpose of proteasome inhibition is to block the degradation of altered proteins performed by this complex, stimulating the formation of a toxic environment which ultimately triggers the apoptotic processes. As a matter of fact, the use of proteasome inhibitors (PI) in oncology is constantly gaining more interest, as emphasized by the development of next generation proteasome inhibitors. Some of them has already entered clinical trials also in solid malignancies providing outstanding outcomes in terms of overall survival (OS) 15 . In this framework, the clinical success of Bortezomib in treatment of haematological disorders, in particular multiple myeloma, has pioneristically validated the relevance of this therapeutic strategy. Bortezomib is a dipeptidyl boronic acid reversible inhibitor of the chymotrypsin-like activity of the 20S proteasome 14,20,21 . Bortezomib was rst synthesized in 1995 and subsequently approved in 2003 as a preferential treatment of multiple myeloma 22 . Bortezomib has been reported to show a potent anticancer activity, both in vitro and in vivo, also in other cancer cell lines, like prostate cancer, pancreatic cancer, renal cell carcinoma and squamous cell carcinoma [23][24][25][26] . In addition, several clinical trials evaluated the effect of Bortezomib in combination with chemotherapy or other targeted therapies in HNC [27][28][29][30][31][32][33][34] .
The aim of our research was to evaluate the in vitro and in vivo antitumoral activity of Bortezomib in HNC. We analyzed the in vitro effects of Bortezomib on cells proliferation, cells death, cells cycle regulation, apoptosis, autophagy, expression, and activation of several proteins involved in pro-survival signaling pathways in human and mouse HNC cell lines, including salivary gland cancer cell lines. We also evaluated the effects of Bortezomib in inhibiting the proteasome activity in individual HNC cell lines and correlated for the rst time this effect with the modulation of different signal transduction pathways involved in head and neck neoplastic transformation. In addition, we explored for the rst time the in vivo effects of Bortezomib in mice transplanted with murine adenocarcinoma salivary gland SALTO-5 cell line.
Our study therefore provides additional information on the antitumoral effect of Bortezomib and on the development of resistance mechanisms associated with the activation of deregulated speci c signaling pathways in HNC cell lines.

Trypan Blue Exclusion Assay
For trypan blue exclusion assay, cells were seeded at 5 x 10 4 /well in 24-well plates and incubated at 37°C to allow cells attachment. After 24 hours, the medium was changed and cells were incubated for 24, 48, and 72 hours with Bortezomib (6.25-100 nM) or DMSO. Thereafter, adherent as well as suspended cells of each well were harvested and stained with trypan blue (Sigma-Aldrich, Milan, Italy) and counted under a light optical microscope 99 . The experiments were repeated three times, and the percentage of dead cells was compared with the total number of cells 100 .

FACS analysis
Asynchronized log-phase growing cells (60% con uent, approximately 2.5 x 10 5 cells/well in 6-well plates) were treated with Bortezomib or with DMSO in a complete culture medium. Z-VAD-FMK was used at a nal concentration of 40 µM for 2 hours before addition of Bortezomib. After 48 hours, adherent and suspended cells were harvested, centrifuged at 1500 rpm for 10 minutes and washed twice with cold phosphate buffered saline (PBS). The assay was then performed as previously described 101 . Cells were analyzed by ow cytometry using a FACS-Calibur cytometer running CellQuest Pro 5.2 software (BD Biosciences, San Jose, CA, USA).

Western Blotting
About 1 x 10 6 cells were seeded in 100 mm tissue culture dishes 24 hours prior to the addition of 12.5 (SCC-15 and CAL-27) or 25 nM (FaDu, A-253, SALTO-5) of Bortezomib or the vehicle control. After 24 hours (for SCC-15) and 48 hours of treatment, cells were harvested, washed twice with cold PBS and lysed in RIPA buffer as previously described 102 . For immunoblotting analysis, 15-80 µg of cell lysates (depending on the experimental setting) were resolved in 10% SDS-PAGE and then transferred to nitrocellulose membranes 103 . Equal loading and transfer of proteins was veri ed by Ponceau red staining of the membranes and by analyzing actin expression. The assay was then performed as previously described 72 . Native Gel Electrophoresis Crude cell extracts (e.g., soluble fraction of the cell cytosol) were extracted under non-denaturing conditions through freeze-thawing cycles in 250 mM sucrose, 20% glycerol, 25 mM Tris-HCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM DTT, 2 mM ATP, pH 7.4. Thereafter, lysates were cleared by centrifugation at 13000 rpm, 20 minutes, 4°C and the protein concentration was determined by Bradford assay. For each experimental condition, 75 µg of proteins were separated under native conditions employing 3.5 % acrylamide gel. Gels were then harvested in a clean dish and soaked in the reaction buffer (50 mM Tris, 5 mM MgCl 2 , 1 mM ATP, pH 7.5), which had been supplemented with 75 µM 7-amino-4-methylcoumarin (AMC) labeled Suc -Leu -Leu -Val -Tyr -AMC peptide (referred to as LLVY-AMC) (Boston Biochem, Boston, USA), a highly speci c uorogenic substrate of the proteasome chymotrypsin-like activity. This enzymatic proteolytic activity, which has been proven to be linearly correlated with the light intensity, was then recorded through a gel-documentation system (excitation λ = 365 nm; emission λ = visible) 104 . Proteins were then transferred to a HyBond-ECL nitrocellulose lters and probed with an antibody which recognizes an epitope shared by α1-7 subunits, but not by α4 (hereafter referred to as pan-α-subunits) (Protein-tech Group, Manchester, UK), diluted 1:3000 in 0.02% Tween-PBS fat-free milk, and then incubated with a Horseradish Peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody (Biorad, Hercules, CA, USA), diluted 1:50000 in 0.2% Tween-PBS fat-free milk.

Treatment of BALB-neuT mice with Bortezomib
Transgenic BALB-neuT male mice were mated with BALB/c females (H-2 d ; Charles River, Calco, Italy) in the animal facilities of Tor Vergata University. Founder male BALB-neuT mice were kindly provided by Prof. G. Forni and Prof. F. Cavallo (University of Torino, Italy) 105 . Progenies were con rmed for the presence of the transgene by PCR 106 . BALB-neuT mice were subcutaneously injected in the right ank with a 0.2 ml suspension containing 1 x 10 6 SALTO-5 cells in phosphate-buffered saline (PBS). Groups of BALB-neuT mice (8 mice per group) were treated i.p. with Bortezomib (0.5 mg/kg in 400 µl PBS + DMSO, twice a week) or with vehicle only (400 µl PBS + DMSO, twice a week) one week after the SALTO-5 tumor challenge. Mice were sacri ced at the rst signs of distress 107

Analysis of antitumor activity in vivo
Tumor growth was monitored weekly until tumor-bearing mice were sacri ced when the tumor exceeded a 20 mm width by cervical dislocation. Tumors were measured by a caliper in two perpendicular dimensions, and the volumes were calculated using the formula: width 2 x length/2 100, 108 .
Histological analysis of tumors from mice treated with Bortezomib by optical microscopy Tumors from three animals from each group of mice were used for histological examination after hematoxylin/eosin staining using 3 µm thick para n sections. Necrotic areas were measured using ImageJ software on 10 microscopic elds. Immunohistochemistry (IHC) was used to analyze the presence of caspase 3-positive cells (apoptotic cells) and the expression of ErbB2, AKT and phospho-AKT in tumors from DMSO-and Bortezomib-treated mice 109 . For IHC, antigen retrieval was performed on 3 µm thick para n sections using EDTA citrate, pH 7.8, or citrate pH 6.0 buffers for 30 minutes at 98°C. Sections were then incubated for 1 hour at room temperature (anti-cleaved caspase 3 and anti-ErbB2 antibodies) or overnight at 4°C (anti-AKT and anti-phospho-AKT) with primary antibodies. To remove nonspeci c binding, slides were washed using PBS/Tween 20, pH 7.6. Antibody-antigen binding was revealed by the Horseradish Peroxidase-3,3-diaminobenzidine (HRP-DAB) Detection Kit (UCS Diagnostic, Rome, Italy). The count of cleaved caspase 3-positive cells (apoptotic cells) was performed on 10 microscopic elds at 200x magni cation. Semiquantitative ErbB2, AKT and phospho-AKT expression in tumors derived from DMSO-and Bortezomib-treated mice was estimated at x200 magni cation in at least 10 elds by two investigators in a blind fashion. ErbB2, AKT and phospho-AKT expression levels (negative, 0; weakly positive, 1; moderately positive, 2; strongly positive, 3) were scored. The interobserver reproducibility was > 95%. Sections were observed and photographed by Olympus BX53 light microscope 101,108,110 . Transmission electron microscopy Ultrastructural analysis was performed on SALTO-5 cells treated with Bortezomib (12.5 nM for 24 hours) or with DMSO. After treatment, cells were xed in 2.5% glutaraldehyde in PBS pH 7.4, and the samples were processed for ultrastructural analysis following routine procedures and observed by a Morgagni 268D transmission electron microscopy 111 .

Statistical analysis
The percentage of cells survival, different phases of the cell cycle and of cells death were preliminarily veri ed using the Kolmogorov-Smirnov test, and the data sets were analyzed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. Differences in the intensity of immunoreactive bands were evaluated by a two-tailed Student's t-test or one-way ANOVA followed by Tukey's post-hoc signi cance test. Values with p ≤ 0.05 were considered signi cant. Survival curves and tumor volumes were analyzed using the Kaplan-Meier method and compared with a log-rank test (Mantel-Cox). Differences in tumor volumes were regarded as signi cant when the p-value was ≤ 0.05. The effect of Bortezomib on cells proliferation was dose-and time-dependent as compared to control cells. The greatest effect was obtained at the concentrations of 25, 50 and 100 nM and after 48 and 72 hours of treatment (Fig. 1). SCC-15 were the most sensitive cells to the drug's effect.

Results
In addition, the concentration of Bortezomib that inhibited 50% of cells growth (IC 50 ) after 48 and 72 hours was also determined. FaDu cells were the most resistant to Bortezomib activity, while SCC-15 was the most sensitive cell line (Table 1).  induced, at all doses tested, a signi cant increase in A-253 cells for the percentage of cells in the G2/M phase ( Table 2).
To con rm the induction of apoptosis, cells were simultaneously treated with Bortezomib at the highest dose and with the universal caspase inhibitor, Z-VAD-FMK. Administration of this drug was able to signi cantly reduce the number of cells in the subG1 phase as compared to the single treatment with Bortezomib at the highest dose, thus suggesting the induction of cell death by apoptosis following treatment with Bortezomib in HNC cell lines (Table 2).  In accordance with a general inhibition of proteasome activity, the poly-ubiquitinated proteins, (i.e., the natural substrates of capped proteasome assemblies), which were assayed by denaturing and reducing Western blotting, turned out to be signi cantly increased in the presence of Bortezomib under all experimental conditions (Supplementary Fig. S2).
To further validate their identity, proteasome particles were then transferred to a nitrocellulose lter and probed with an antibody which recognizes all catalytic assemblies of proteasome (i.e., 30S, 26S, and 20S), since it is raised against a peptide covering residues shared by the α1-7 subunits of 20S proteasome, with the exception of α4 (hereafter referred to as pan-α-subunits). By probing the lters some unexpected ndings were observed (Fig. 6a,  showed the appearance of non-canonical proteasome assemblies with an apparent mass/charge ratio very similar to that reported by other authors in a similar experimental condition 41 (Fig. 6a, bottom panel, red arrow). It is worth pointing out that SCC-15 cells displayed a decrease in overall proteasome content also in control cells harvested after 14 hours (with respect to control cells harvested after 7 hours), thus con rming the observed loss of proteasome activity (Fig. 6). Conversely, SALTO-5 cells appeared to be somewhat resistant to this phenomenon, since this cell line did not display any signi cant decrease of proteasome particles content (Fig. 6a, bottom panel, and Fig. 6b) with the exception of cells treated with 50 nM for 12 hours.
In addition, A-253 and CAL-27 cells displayed a very high 20S/capped assemblies (30S + 26S) ratio, even though the pattern of activity (Fig. 6a, bottom panel, and Fig. 6b) was comparable to that of other cell lines analyzed which showed a more balanced 20S/capped assemblies ratio.
To better address these ndings, the same crude cell extracts, run by native gel electrophoresis, were analyzed by denaturing, and reducing Western blotting. Interestingly, the content of free α7 and Rpt5 subunits, which are representative of the 20S and 19S particles, respectively, ranged from unchanged or even increased under the investigated experimental conditions (Supplementary Fig. S2).
To further verify the observed phenomenon, FaDu cells, which displayed the apparent highest extent of proteasome loss, were further stimulated with 25 and 50 nM Bortezomib for 30 minutes and 2 hours (Fig. 7). Proteasome activity was almost null as early as after 30 minutes of stimulation in the presence of 50 nM Bortezomib, whilst a residual activity was observed in the presence of 25 nM Bortezomib (Fig. 7, left panel). After 2 hours of stimulation, the proteolytic activity was undetectable in the presence of all Bortezomib concentrations. Interestingly, by probing the lter with the anti-pan-α subunits antibody, it was observed that the proteasome assemblies were unaffected by Bortezomib after 30 minutes of stimulation (Fig. 7, left panel). Conversely, after 2 hours of Bortezomib treatment, a very robust loss of proteasome assemblies was observed in the presence of 50 nM Bortezomib, whereas residual proteasome assemblies were detected in the presence of 25 nM Bortezomib (Fig. 7, right panel).

Bortezomib inhibited the in vivo growth of SALTO-5 cells transplanted in BALB-neuT mice
Treatment with Bortezomib signi cantly affected tumor growth in vivo. BALB-neuT mice were subcutaneously injected in the right ank with SALTO-5 cells and treated i.p. with Bortezomib. Tumor volumes in the Bortezomib-treated mice were signi cantly less than those in the control-treated mice after 3 weeks (53.1 vs 90.5 mm 3 ; p = 0.030). This signi cant difference was maintained until the 5th week, when two control-treated mice were sacri ced due to the excessive size of the tumor (251.6 vs 1035 mm 3 ; p = 0.0001). The remaining control-treated mice were sacri ced at 6 (5 mice) and 7 (1 mouse) weeks ( Fig. 8a). At this stage (7 weeks after tumor challenge) only one Bortezomib-treated mouse was sacri ced due to excessive size of the tumor. In contrast, the remaining Bortezomib-treated mice were sacri ced at week 9 (3 mice), 10 (2 mice), 11 (1 mouse) and 12 (1 mouse). The mean survival signi cantly increased for the mice treated with Bortezomib, as compared to the control-treated mice (9.5 vs 6 weeks, Bortezomib-treated mice vs control-treated mice; p = 0.0001) (Fig. 8b), indicating that the risk of SALTO-5 cell growth in control-treated mice was 22.57 times greater than that of mice treated with Bortezomib (Table 3). Our results demonstrated that treatment with Bortezomib interfered with the in vivo tumor growth of transplanted salivary gland cancer cells SALTO-5.  (Fig. 9).
The presence of apoptotic cells was evaluated by the expression of cleaved caspase 3 in cancer cells employing immunohistochemical analysis (Fig. 9). The number of apoptotic cells within the tumors from Bortezomib-treated mice (62.8 ± 5.1) was higher than that from control-treated mice (8.4 ± 1.1) (p = 0.0001).
The expression of ErbB2 on tumor cells in vivo was evaluated by immunohistochemical analysis as well (Fig. 9). Tumors from Bortezomib-treated mice showed a signi cantly lower expression of ErbB2 than that from control-treated mice (1.0 ± 0.4 vs 2.3 ± 0.8 staining intensity, p = 0.04). In addition, AKT phosphorylation was signi cantly decreased in tumors from mice treated with Bortezomib, as compared to those treated with DMSO (0.3 ± 0.2 vs 0.8 ± 0.2 staining intensity, p = 0.04). On the other hand, the same treatment did not affect AKT expression in tumors (Fig. 9).

Ultrastructural analysis of SALTO-5 cells in vitro treated with Bortezomib
Ultrastructural analysis was performed by transmission electron microscopy on SALTO-5 cells treated with Bortezomib or DMSO at the concentration of 25 nM for 24 hours. DMSO-treated cells showed heterogeneous forms with a predominance of round over stretched cells. The nuclei appeared large, mainly formed by euchromatin with low dense heterochromatin in the periphery. In the cytoplasm several mitochondria and cisterns of rough endoplasmic reticulum were detected, with few vacuoles (Fig. 10a,b). Conversely, Bortezomib-treated cells showed mainly necrotic features (Fig. 10c) with cytoplasm swelling and the presence of numerous cytoplasmic vacuoles surrounded by a single or double membrane, the latter being probably of autophagic origin (Fig. 10d). Few apoptotic cells were also visible (Fig. 10e).

Discussion
The use of new therapeutic agents is being evaluated in HNC [5][6][7][8] . Among these, inhibitors of the UPS, and mainly of proteasome, are assuming particular importance. The proteasome is a multisubunit complex, which is responsible for the selective proteolysis of damaged, denatured or structurally aberrant proteins 42 . The purpose of proteasome inhibitors is to block the degradation of the altered proteins, performed by this complex, stimulating their building up in intracellular compartments, thus bringing about the formation of a toxic environment and the subsequent need to activate apoptotic processes 43  The pathway of MAP kinases family (Mitogen Activated Protein kinases) is one of the main signal transduction pathways triggered by EGFR and ErbB2. It has been shown that these receptors are often over-expressed and play a role in the carcinogenesis process of HNC 35 . Our results indicated that Bortezomib signi cantly decreased the expression level of EGFR and/or ErbB2 in SCC-15, CAL-27, and A-253 cells but not in FaDu cells. Accordingly, the decreased expression of the ErbB2 receptor only in tongue, salivary gland but not pharynx cell lines is an additional nding which outlines Bortezomibmediated biological effect in a cell line speci c-dependent modality. One study showed that Bortezomib was not able to modulate EGFR expression in HNSCC cell lines 57 . EGFR and ErbB2 are frequently overexpressed in HNC cells 35,68 and are frequently prone to heterodimerization that confers tumor growth advantage 69,70 .
To better elucidate the possible intracellular signaling mechanisms involved in the effects of Bortezomib, we have analyzed the phosphorylation status of ERK1/2, p38 and JNK/SAPK kinases (p46/p54), important members of the serine/threonine MAP kinase family. As a matter of fact, the activation of ERK1/2 can promote proliferation, differentiation, adhesion, migration, and survival but also apoptosis [71][72][73][74] . In addition, the activation of p38 and JNK1/2 stress pathways modulates cell proliferation, differentiation, and apoptosis 75,76 and Bortezomib-induced activation of p38 and JNK is associated with the induction of apoptosis in several types of cancer 77,78 . Our ndings showed that the treatment with and non-small cell lung cancer cells 82 . However, our ndings showed that Bortezomib had no effect on AKT expression and phosphorylation on the pharynx cell line FaDu.
Finally, the e cacy of proteasome inhibition by Bortezomib in vitro was investigated focusing on whether different responses on the modulation of the signaling pathway molecules were dependent on a different sensibility of cells to Bortezomib-induced inhibition of proteasome activity. Only two studies showed that Bortezomib affects proteasome activity by inducing the accumulation of ubiquitylated proteins in larynx 56 and mouth oor cells 59 . Thus, a further novelty of our study is the analysis of the structural and functional effects of Bortezomib on the proteasome assemblies in HNC cell lines. On the other hand, our results uncover some ndings which are worth being further discussed. Although every cell line displayed sensitivity to Bortezomib treatment, some differences were observed. Thus, the different responses observed upon Bortezomib treatment in the HNC cell lines could be due to a different extent of proteasome inhibition. Indeed, FaDu cells, which were reported to be the most resistant to Bortezomib treatment in terms of viability, were those displaying the lowest extent of proteasome inhibition after 12 hours and 24 hours of stimulation, regardless the Bortezomib concentration administered. It is important to know that, as highlighted above, Bortezomib was not able to induce modulation of EGFR, ErbB2, JNK, p38 as well AKT proteins in FaDu cells. The ineffectiveness of Bortezomib in modulating these signal transduction pathways thus parallels the low e cacy of Bortezomib in inhibiting the proteasome activity.
However, the Bortezomib inhibitory effect on overall proteolytic activity was several-fold greater when the proteasome assemblies were harvested and analysed at earlier time-points. Without ruling out the possibility that FaDu cells may have evolved canonical mechanisms of drug resistance (e.g., drug secretion and/or detoxi cation, or selective downregulation of 19S subunits 87 ), the resistance of these cells to Bortezomib-induced apoptosis, which is in sharp contrast with the complete early proteasome inhibition after 2 hours, can be likely explained through two different and not mutually exclusive hypotheses, namely: i) Bortezomib, being a reversible inhibitor, is displaced from the β5 catalytic site at a higher rate than in other cells; ii) among all cells employed in this study, FaDu are those which more readily synthesize de novo proteasome assemblies. Hypothesis i) indeed re ects a chemical property of Bortezomib which contributes to the resistance through which the cells can bypass the drug-induced death. However, although speculative at this stage, we envisage that hypothesis ii) may be of greater relevance to explain the observed behaviour for two main reasons: FaDu cells were the only cells clearly inducing the formation of alternative proteasome assemblies, displaying an electrophoretic pattern (mass/charge) of the species after 12 hours and 24 hours (but not at 20 minutes or 2 hours), which clearly resembles that of non-canonical complexes, such as PA28-20S; thus, the formation of these alternative complexes has been previously proposed as an adaptative response to proteasome inhibition, even though the biological role of these assemblies is still unknown 41 .
FaDu cells showed the greatest extent of proteasome loss during treatment, a phenomenon which, to the best of our knowledge, has never been reported in the presence of a proteasome inhibitor.
Remarkably, this Bortezomib-induced effect was observed, though to a variable extent, in all human cell lines tested so far, underscoring that it may be a general issue of pharmacological relevance, if con rmed in vivo.
For what concerns the proteasome loss, we cannot rule out a priori that it is a technical artifact, even though it seems unlikely, since the immunostaining was performed with an antibody which targets an epitope shared by 6 out of 7 α-subunits of the proteasome; therefore, it looks unlikely that the residues, which are part of the epitope, undergo post-translational modi cations which shield it from antibody recognition in all subunits; upon analysis by denaturing and reducing Western Blotting of the same cytosolic extract that was run by native gel electrophoresis, the immunostaining of individual proteasome subunits revealed no difference at the earliest time-point while at the latest time-point a marked tendency toward increase was observed for all tested cell lines but A-253 cells.
Therefore, this nding envisages that the proteasome loss should not be attributable to the enhanced degradation by macroautophagy (or by other enzymatic pathways) nor to translocation of the particles into other intracellular compartments (e.g., outer surface of ER and nucleus) which are excluded from crude cell extracts preparation. It is then conceivable that Bortezomib inhibition induced a disassembly of proteasome and FaDu cells respond to this insult more quickly than other cell lines, at least in vitro.
Overall Our results showed for the rst time that intraperitoneal administration of Bortezomib (0.5 mg/kg, twice a week) reduced the growth of SALTO-5 murine cells in mice. The increase in mean survival time of the mice treated with Bortezomib was relevant, as compared to that of the control mice. Furthermore, the growth risk of SALTO-5 cells in control mice is over 13-fold higher than that of Bortezomib-treated mice. In addition, the histological examination of tumors from Bortezomib-treated mice showed extensive necrosis and presence of apoptotic cells, as compared to the control mice. According to the in vitro results, the IHC analysis revealed the decrease of the expression of ErbB2 and of the AKT phosphorylation in tumors from Bortezomib-treated mice with respect to control mice. AKT inhibition by    Effect of Bortezomib on the expression and activation of ErbB receptors (EGFR and ErbB2) and signaling transduction pathway molecules. Western blotting analysis was performed on HNC cells treated with Bortezomib (Bor) or DMSO for 24 or 48 hours, at a dose of 12.5 (SCC-15, CAL-27) or 25 nM, as reported in that of total ERK, p38, JNK, and AKT proteins, respectively. The intensity of the bands was quanti ed using the ImageJ software after blot scanning of two independent experiments. The ratios are reported.
Actin was used as an internal control. n.d.: not detectable. Uncropped western blots are reported in Supplementary Informations.    Intensity of ErbB2, AKT and pAKT expression within the tumors was semiquantitative evaluated as described in Materials and Methods, and the results are shown in the adjacent bar graph (#: p=0.0015; *: p=0.04; **: p=0.0001). Scale bars correspond to 100 µm. Figure 10