The use of new therapeutic agents is being evaluated in HNC5–8. 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 proteins42. 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 processes43. Bortezomib ([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid) is a boronic acid derivative which contains pyrazinoic acid, phenylalanine and leucine with boronic acid within its structure. It is a specific and reversible inhibitor of the chymotrypsin-like activity of the 20S proteasome14,20,21. Bortezomib has been reported to possess potent anticancer activity, both in vitro and in vivo, in prostate cancer, pancreatic cancer, renal cell carcinoma, and squamous cell carcinoma23–26,44,45.
Preclinical studies have shown that Bortezomib, when used alone, inhibited cells growth, induced apoptosis, autophagy, and modulation of individual signaling transduction pathways in HNSCC (Head and neck squamous cell carcinoma) cells in vitro46–56. However, none of these studies have analyzed the simultaneous effects of Bortezomib on proteasome, apoptosis, autophagy and signaling transduction pathways involved in cellular transformation in HNC cell lines. Thus, one of the novelties of our study is the detailed analysis of the correlation between inhibition of the proteasome and activation of apoptosis, autophagy and modulation of cell survival signaling transduction pathways, that is a concern which no previous single study has been dealing with. Bortezomib was previously shown to activate apoptosis by modulating the expression of pro-apoptotic and anti-apoptotic proteins48,49 following the activation of caspases and/or the cleavage of PARP-1 protein46,48,50–52,57 and hypodiploidia and phosphatidylserine externalization in HNSCC cell lines46,48,56. Bortezomib was shown to stimulate autophagy in HNSCC cells in two studies showing autophagosomes formation, upregulation of LC3-I, -II, Beclin-1 and the JNK-dependent phosphorylation of Bcl-2 after bortezomib treatment58,59. It was also suggested that autophagy attenuated the Bortezomib cytotoxicity59. Bortezomib was able to modulate expression and activation of signaling pathways in HNSCC. Two studies demonstrated Bortezomib inhibition of AKT activation and mTOR in HNSCC cell lines46,60. Other studies demonstrated the upregulation of STAT3 after Bortezomib treatment and the anti-tumoral efficacy of a co-treatment with STAT3 inhibitors in HNSCC cells55. Only two studies showed that Bortezomib affects proteasome activity by inducing the accumulation of ubiquitylated proteins in larynx (UM-SCC-11A, -11-B) [56] and mouth floor (SCC1) cell lines59. It is worth pointing out that the majority of these studies investigated the effect of Bortezomib in the modulation of individual biological pathways being restricted to SCC cell lines arising in head and neck. In our study the in vitro and in vivo effects of Bortezomib were for the first time analyzed in a salivary gland adenocarcinoma experimental model. Salivary gland carcinomas represent 6–8% of HNC, with heterogeneous morphologies and clinical outcomes3. The mainstream therapy for these types of cancer, when feasible, is the surgery followed by radiation therapy. The chemotherapy has demonstrated mixed results with low responses for advanced or metastatic malignant tumors, so that new targeted therapies are under evaluation61–63. Bortezomib has been evaluated for the treatment of only adenoid cystic carcinoma in combination with doxorubicin in a phase II trial, leading to no complete or partial responses in patients32.
We demonstrated that Bortezomib inhibited in a dose- and time-dependent manner the growth and induced cell death of SCC cells from tongue (SCC-15 and CAL-27), pharynx (FADU) and of salivary gland adenocarcinoma (A-253). The tongue cell line SCC-15 turned out to be the most sensitive one to the effect of the drug, whereas the pharyngeal carcinoma cell line (FaDu) was the most resistant to the action of Bortezomib.
We demonstrated that treatment with Bortezomib significantly increased the percentage of cells in the subG1 phase, which is conventionally associated with apoptotic phenomena. The use of the inhibitor Z-VAD-FMK further confirms the induction of cell death by apoptosis following treatment with Bortezomib in these cell lines. In addition, Bortezomib induced a significant increase in the percentage of cells in the G2/M phase at all doses in the A-253 cell line, thus suggesting the induction of apoptosis and a cell cycle block in the G2/M phase. It is worth remarking that only the salivary gland adenocarcinoma A-253 cell line showed a G2/M arrest simultaneous to apoptosis, thus indicating a different response to the drug. This cell cycle pattern after Bortezomib treatment was previously reported in tumor cell lines from larynx cancer56, colorectal cancer64, non-small cell lung carcinoma65, prostate cancer43, Ewing's sarcoma66, malignant mesothelioma and breast cancer67.
Our findings of apoptosis activation by Bortezomib corroborated previous results employing HNSCC cell lines46–52 and showed for the first time Bortezomib-mediated apoptosis in a salivary gland adenocarcinoma cell line (A-253).
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 HNC35. Our results indicated that Bortezomib significantly 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 finding which outlines Bortezomib-mediated biological effect in a cell line specific-dependent modality. One study showed that Bortezomib was not able to modulate EGFR expression in HNSCC cell lines57. EGFR and ErbB2 are frequently over-expressed in HNC cells35,68 and are frequently prone to heterodimerization that confers tumor growth advantage69,70.
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 apoptosis71–74. In addition, the activation of p38 and JNK1/2 stress pathways modulates cell proliferation, differentiation, and apoptosis75,76 and Bortezomib-induced activation of p38 and JNK is associated with the induction of apoptosis in several types of cancer77,78. Our findings 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 finding corroborated other studies in which it has been shown that Bortezomib induced apoptosis by activating JNK kinase79 in multiple myeloma80, 81 and in non-small cell lung cancer82. Our finding 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 lines47,58. Accordingly, our results showed that Bortezomib induced autophagy in human HNSCC cells, but the process was then blocked, as showed by the increase of p6239. The same effect was observed for the first time in the salivary gland adenocarcinoma cell line. The block of the autophagic flux by Bortezomib was reported in ovarian cancer cells, hepatocellular carcinoma cells and endometrial cancer cells83, breast cancer cells84, and B-Raf-mutated melanoma cells85.
Furthermore, we evaluated the in vitro effect of Bortezomib on the activation of the serine/threonine kinase AKT, which plays a role in various physiological processes, such as differentiation and cell cycle, transcription and translation, metabolism, and apoptosis. AKT activation, which occurs through phosphorylation of serine 273, depends on the activation of phosphatidyl inositol 3 kinase (PI3K) and triggers a cell survival signal86. Our results showed that treatment with Bortezomib results in the inhibition of AKT phosphorylation in both the tongue squamous carcinoma cell lines (SCC-15, CAL-27) and in the salivary gland adenocarcinoma cell line (A-253 cells) which was accompanied by a significant decrease in the expression of the total form of AKT in these cell lines. The inhibition of AKT phosphorylation by Bortezomib is a key molecular event for Bortezomib-mediated apoptosis in HNC46, 60 and non-small cell lung cancer cells82. However, our findings showed that Bortezomib had no effect on AKT expression and phosphorylation on the pharynx cell line FaDu.
Finally, the efficacy 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 larynx56 and mouth floor cells59. 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 findings 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 efficacy 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 detoxification, or selective downregulation of 19S subunits87), 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 reflects 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:
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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 unknown41.
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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 confirmed 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
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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 modifications which shield it from antibody recognition in all subunits;
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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 finding 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 in vitro findings suggest that in HNC, showing limited proteasome resistance to Bortezomib and simultaneous upregulation of ErbB receptors-mediated signaling, anti-ErbB receptors antibodies or inhibitors of the ErbB receptors intrinsic tyrosine kinase activity should be used in combination with proteasome inhibitors.
In addition, regarding the analysis of the effects of Bortezomib on experimental models in HNC, there are several studies which have shown that Bortezomib has promising anticancer activities in mouse tumor models45,46,56,88−90. However, only two studies analyzed the ability of Bortezomib to counteract the in vivo HNC tumor growth, but they were restricted to xenografts implanted human larynx or tongue squamous cell carcinoma cell lines46,56. Accordingly, none of them have investigated the in vivo effect of Bortezomib in salivary gland carcinoma cell line. Thus, this is the first study showing the in vitro and in vivo growth inhibitory properties of Bortezomib in a salivary gland carcinoma cell line (SALTO-5) and the in vivo effects of Bortezomib on ErbB2, AKT and caspase 3 expression in SALTO-5 cell line transplanted in BALB-neuT mice. The Bortezomib effects on SALTO-5 cells were first analyzed in vitro. We observed apoptosis and inhibition of ErbB2, p38 and AKT, and activation of JNK in Bortezomib-treated SALTO-5 cell line. In contrast to the human cells analyzed, Bortezomib induced an increase of ERK2 phosphorylation in SALTO-5 cells, which was still associated with the activation of the apoptotic process. On the other hand, regarding the effect of Bortezomib on proteasome, SALTO-5 cells displayed a significant recovery of proteasome particles after 24 hours and of proteasome subunits content associated to a decrease of poly-ubiquitinated proteins. Although the study of apoptosis pathway did not put in evidence any resistance of these cells greater than that of SCC-15, A-253 or CAL-27 cells, it is likely that this mechanisms of resistance to the drug may emerge over a prolonged time of treatment. Cytotoxic and apoptotic effects of Bortezomib in SALTO-5 cell line were also observed by ultrastructural analysis.
In light of the in vitro results, we also evaluated the in vivo anti-tumor effects of Bortezomib on tumor growth in BALB-neuT mice subcutaneously inoculated with syngeneic murine SALTO-5 cells. Several studies have also evaluated the efficacy of Bortezomib in relation to the route of administration, and it has been shown that intraperitoneal (i.p.) administration, at least twice a week, resulted in greater Bortezomib activity with less toxicity91. These preclinical investigations have collectively demonstrated the anticancer activity of Bortezomib when used as monotherapy or in combination with chemotherapy, radiotherapy, or other anti-neoplastic agents91–96.
Our results showed for the first 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 Bortezomib in vivo was observed in homogenates from tumors of a tongue squamous cell carcinoma cell line transplanted in mice46. Our results provide evidence that Bortezomib inhibited in vivo the expression of ErbB2 simultaneously to that of AKT. One previous study showed that tumor specimens, from mice transplanted with a human larynx cell line and treated with Bortezomib, displayed cell nuclear condensation and tissue degradation, as well as apoptotic areas56.
Overall, our results showed that anti-cancer activities of Bortezomib in tongue, pharynx and salivary gland cancer cells were dependent on cell line histotype and associated with the different extent of proteasome inhibition. The inhibition of proteasome was in turn associated with the modulation of the main signaling transduction pathways involved in HNC cellular transformation. Furthermore, for the first time we showed that Bortezomib displayed in vitro and an in vivo antitumor activity in an adenocarcinoma of the salivary gland. Intraperitoneal administration of Bortezomib interfered with the in vivo tumor growth of SALTO-5 cells in BALB-neuT mice and prolonged mice median survival time. The inhibition of tumor growth by Bortezomib was associated with tumor necrosis and apoptosis and with the simultaneous inhibition of ErbB2, AKT.
Our in vitro and in vivo findings further support the use of the proteasome inhibitor Bortezomib for the treatment of HNSCC and adenocarcinomas of the salivary gland and reveal its ineffectiveness in counteracting the activation of deregulated specific signaling pathways in HNC cell lines when resistance to proteasome inhibition is developed, thus suggesting the combined use of Bortezomib and specific drugs targeting signaling transduction pathways unaffected by Bortezomib treatment.