Apoptosis Induction and Gene Expression Profile Alterations of Cutaneous T-Cell Lymphoma Cells following Their Exposure to Bortezomib and Methotrexate

Mycosis fungoides (MF) and its leukemic variant Sézary syndrome (SS) comprise the majority of CTCL, a heterogenous group of non-Hodgkins lymphomas involving the skin. The CTCL’s resistance to chemotherapy and the lack of full understanding of their pathogenesis request further investigation. With the view of a more targeted therapy, we evaluated in vitro the effectiveness of bortezomib and methotrexate, as well as their combination in CTCL cell lines, regarding apoptosis induction. Our data are of clinical value and indicate that the bortezomib/methotrexate combinational therapy has an inferior impact on the apoptosis of CTCL compared to monotherapy, with bortezomib presenting as the most efficient treatment option for SS and methotrexate for MF. Using PCR arrays technology, we also investigated the alterations in the expression profile of genes related to DNA repair pathways in CTCL cell lines after treatment with bortezomib or methotrexate. We found that both agents, but mostly bortezomib, significantly deregulate a large number of genes in SS and MF cell lines, suggesting another pathway through which these agents could induce apoptosis in CTCL. Finally, we show that SS and MF respond differently to treatment, verifying their distinct nature and further emphasizing the need for discrete treatment approaches.


Introduction
Cutaneous T-cell lymphomas (CTCLs) are rare skin malignancies, comprising a heterogeneous group of non-Hodgkin lymphomas derived from skin-homing mature T-cells [1]. Mycosis fungoides (MF) and Sézary syndrome (SS) are considered as the commonest CTCL PLOS ONE | DOI: 10 types and together account for approximately 50% of all CTCL cases [2,3]. MF is considered as the commonest type of CTCL and is initially characterized by patches and infiltrated plaques on the skin which eventually evolve into tumors [4,5]. SS is the leukemic variant of MF and is characterized by erythroderma, lymphadenopathy and the presence of a malignant Tcell clone in the peripheral blood and the skin [2,5]. Advances in therapy for CTCL are mostly focused on the development of novel pharmacological targets but in most cases the response is short and the survival rate is not significantly improved [2,4,6,7]. Combinational therapy of known pharmaceutical agents is another treatment option for CTCL patients, which could be beneficial and lead to better responses compared to monotherapy [8].
Bortezomib, a dipeptide boronic acid analog, is a proteasome inhibitor with antitumor activity [9,10], which reversibly inhibits the chymotryptic activity of the 20S subunit of the proteasome and leads to several downstream effects, including activation of p53, inhibition of NF-kB and accumulation of pro-apoptotic proteins [11]. Bortezomib was approved in 2003 by the FDA for the treatment of multiple myeloma and for relapsed or refractory mantle cell lymphoma [12,13] and a vast number of reports and clinical trials reveal that it can also be used for the treatment of solid tumors, alone or in combination [14][15][16]. Bortezomib has shown promising results in patients with relapsed or refractory CTCL [17][18][19][20]. Nevertheless, its exact molecular mechanism of action in CTCL is not fully understood [21].
Methotrexate is an antimetabolite, a structural analogue of folic acid, which acts as an inhibitor of the enzyme dihydrofolate reductase (DHFR), leading to the depletion of tetrahydrofolate cofactors that are required for DNA and RNA synthesis [22], and therefore to the induction of cell death by secondary genotoxic effects or apoptosis [23]. Methotrexate is an essential anticancer agent particularly for human leukemia, severe psoriasis and the treatment of some solid tumors [24,25] and is already used for the treatment of CTCL patients, alone or in combination with other agents [26][27][28][29][30][31].
We investigated the ability of bortezomib and methotrexate to induce apoptotic cell death in CTCL cell lines, alone or in combination, in order to evaluate each agent's effectiveness in overcoming the denoted CTCLs' apoptotic resistance and determine whether or not combinational therapy presents a higher apoptotic efficiency compared to monotherapy. We further investigated the alterations in the expression profile of selected genes involved in the DNA repair signalling in CTCL cell lines after treatment with bortezomib or methotrexate, aiming at a better understanding of their pathogenesis and the mechanisms of action of the aforementioned pharmaceutical agents in CTCL.

Cell lines and culture
Human CTCL cell lines Hut-78, SeAx and Myla were a generous gift from Dr Margarita Sánchez-Beato (Health Research Institute, Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain). Cells were cultured in RPMI 1640, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cell lines were treated with bortezomib (10nmol/L), methotrexate (10μM) and their combination (bortezomib 10nmol/L and methotrexate 10μM), at 37˚C in a humidified atmosphere with 5% CO 2 , for 24 hours.

Flow cytometric analysis of cell apoptosis
Hut-78 (SS), SeAx (SS) and Myla (MF) cells were cultured with or without addition of the drugs at the indicated concentrations for 24h. Apoptosis was analyzed using the Annexin V/PI assay, as previously described [32].

Human DNA repair signalling pathway detection by RT 2 Profiler PCR Arrays
Total RNA extraction and purification from untreated and drug-treated CTCL cell lines was performed using the RNeasy MinElute kit (Qiagen Ltd., Hilden, Germany), according to the manufacturer's instructions. DNase enzyme digestion was performed to exclude genomic DNA contamination. RNA was quantified using NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, USA). Total RNA with 260/280 ratio of 2.0 or more was utilized for realtime PCR quantification by RT 2 Profiler TM PCR arrays (SABiosciences Corp.) Real-time PCR quantification was performed on a Corbett Rotor Gene 6000 system (Qiagen Ltd.) using an RT 2 Profiler custom PCR Array consisting of a panel of 41 genes involved in DNA Damage Signaling and DNA Repair mechanisms, including two "housekeeping genes". cDNAs were synthesized from 0.8 μgr of total RNA using the commercial RT 2 First Strand Kit (Qiagen, Ltd.), according to the manufacturer's instructions. The obtained cDNA was mixed with RT 2 qPCR master-mix, containing SYBR green (SA Biosciences). The mixture was subsequently added into each well of the RT 2 Profiler custom PCR Array and qPCR was performed according to the manufacturer's instructions. All runs were performed in duplicate. Data were analyzed using the integrated web-based automated software for RT 2 Profiler PCR Array Data analysis (RT 2 Profiler PCR Array Data analysis version 3.5, GeneGlobe Data Analysis) available through SABiosciencies. Fold changes in each gene expression were calculated using the ΔΔ C t method and housekeeping gene controls were used for normalization of the results. A two-fold or greater change in expression was considered significant. Fold-change values less than one were indicative of down-regulation of the gene expression and fold-change values greater than one were indicative of an up-regulation.

Statistical analysis
Statistical analyses were performed with SPSS software (version 16.0; SPSS, Inc., Chicago, IL., USA). All experiments were performed three times in each individual sample and the results were given as the mean value of the three. One-way Anova and LSD/ Bonferroni methods were used to compare the means among more than two different groups. Descriptive statistics, such as mean values and SD, were calculated. Data were reported as mean ± standard deviation (SD) of the mean. A two-sided p value <0.05 was considered statistically significant.

Bortezomib, methotrexate, and their combination induce apoptosis in SS cell lines
We initially analyzed the apoptotic effects of bortezomib and/or methotrexate on the SSderived Hut-78 and SeAx cells (Fig 1). We found that after 24h of drug exposure, Hut-78 cells responded with enhanced apoptosis when treated with either agent or their combination, compared to untreated cells. The higher apoptotic effect was observed after treatment with bortezomib, followed by the two drugs' combination (11.76% and 7.61% vs 1.53% respectively, p = 0.000) and methotrexate (3.98% vs 1.53%, p = 0.000) (Fig 1A and 1D), (data shown as mean ± SD derived from three independent experiments). SeAx cells presented a similar apoptotic profile to Hut-78 cells, exhibiting enhanced apoptotic rates after all treatments, with bortezomib showing the higher apoptotic rate, followed by the two agents' combination and methotrexate (33.0%, 19.48% and 15.31% vs 4.45, respectively p = 0.000) (Fig 1B and 1E).

Bortezomib and methotrexate but not their combination induce apoptosis in the MF cell line MyLa
The MF-derived cell line MyLa was treated with bortezomib, methotrexate or their combination for 24h. FACS analysis after Annexin V/PI assay (Fig 1C and 1F) revealed that both agents enhanced the apoptosis of MyLa cells compared to control, unlike combinational treatment where no statistically significant change was observed. Between the two drugs, methotrexate had the higher apoptotic impact on MyLa cells (3.09% and 1.4% vs 0.44% respectively, p = 0.000 and p<0.01).

Bortezomib-treatment significantly affects DNA repair signaling in SS cell lines
To identify the potential effect of bortezomib on the cellular pathways related to DNA damage and repair of SS cell lines (Hut-78 and SeAx), differences in the mRNA levels of selected genes were examined using a custom RT 2 Profiler PCR array by comparing bortezomib-treated cells with control (untreated) cells. We found that bortezomib had a great effect in the expression profile of genes involved in all DNA repair pathways examined, in both Hut-78 (Table 1) and SeAx ( Table 2) cells, with Hut-78 being affected the most (Figs 2 and 3). Specifically, in Hut-78 cell line, three genes, ERCC6, POLL and XRCC3 were up-regulated more than 2-fold after treatment, with ERCC6 presenting the most significant change (higher than 50-fold) (Fig 2A-2C). ERCC6 was also up-regulated more than 2-fold in SeAx cells, but in a rather moderate way compared to Hut-78, along with CDK7 gene (Fig 3A-3C). In both cell lines, we observed a significant down-regulation of a large number of genes. A total of 25 genes were more than 2-fold down-regulated in Hut-78 compared to untreated cells ( Methotrexate-treatment leads to DNA repair signaling deregulation in SS cell lines PCR array analysis after methotrexate-treatment in Hut-78 and SeAx cells revealed that methotrexate had a significant but lower, compared to bortezomib, impact on the DNA repair related genes' expression (Figs 2 and 3), (Tables 1 and 2). Specifically, in Hut-78 cells (Fig 2A, 2B and 2D), the expression of one gene, ERCC6, was significantly up-regulated (more than 30-fold change), while 7 genes were more than 2-fold down-regulated compared to untreated cells (DSB: XRCC6, XRCC4, PRKDC, RAD50; NER: RAD23A, CDK7 and SSBR related ERCC1). ERCC1 gene's expression presented the highest down-regulation, reaching a more than 600-fold change compared to untreated cells. Same wise, in SeAx cells the expression of several genes was affected after methotrexate treatment (Fig 3A, 3B and 3D). Six genes were up-regulated

Bortezomib-treatment effect on DNA repair signaling in MyLa cell line (MF)
Bortezomib-treatment of MyLa cell line followed by PCR arrays analysis revealed an altered expression profile of genes involved in the DNA repair pathways, compared to untreated cells (Fig 4A-4C), (Table 3) (Table 3), compared to untreated MyLa cells. LIG1 and XRCC2 genes showed the highest expression change, a more than 4-fold downregulation.

Methotrexate-treatment effect on DNA repair signaling in MyLa cell line (MF)
Methotrexate-treatment of MyLa cell line had a rather moderate effect on the expression profile of the selected DNA repair related genes, compared to untreated cells, as revealed through PCR arrays analysis (Fig 4A, 4B and 4D). We found that only a total of 4 genes were significantly affected (Table 3). Specifically, ATR, BRCA1 and RAD51 genes were up-regulated more than 2-fold, while the expression of RAD23A (NER mechanism) was down-regulated.

Discussion
Although many chemotherapeutic agents have been used in the treatment of MF/SS, no single therapy has shown superior or efficient effect in the long term, especially for refractory, extensive and/or advanced disease, which are mostly approached through systemic treatments that also fail to provide a permanent therapeutic solution [33]. Since there are no current curative treatment options for CTCL patients, there is an even higher necessity for a better insight into the pathogenesis of the disease. We investigated for the first time whether two well-known therapeutic agents, bortezomib and methotrexate, interfere with the DNA repair mechanisms in CTCL cell lines and manage to overcome their resistance to apoptosis.
We initially tested the ability of the aforementioned agents and their combination to induce apoptosis in SS (Hut-78 and SeAx) and MF (MyLa) cell lines. Our data demonstrate that both SS cell lines (Hut-78 and SeAx) respond with statistically enhanced apoptosis after all three treatments, with bortezomib presenting the highest effect. The MF cell line MyLa responded with a statistically significant but rather moderate augmentation in apoptosis only after treatment with each agent separately and not their combination, with methotrexate presenting the higher apoptotic impact between the two. Our results are in accordance with previously published studies, since both agents have been reported to induce apoptosis in CTCL through various mechanisms. Specifically, methotrexate has been shown to induce apoptosis in MF/SS cell lines through the enhancement of FAS expression in cell lines and leukemic cells by inhibiting methylation of the FAS promoter and thereby de-repressing gene expression, resulting in the upregulation of proteins essential to T-cell apoptosis [34,35]. Bortezomib has also been reported to induce apoptosis in CTCL cell lines. Previous studies have shown that bortezomib induces nuclear translocation of IkBa resulting in gene-specific suppression of NF-kB-dependent transcription and induction of apoptosis in CTCL [36,37]. Moreover, bortezomib induces CTCL cell apoptosis by inhibiting the expression of the antiapoptotic genes cIAP1 and cIAP2 and the proto-oncogene Bcl3 [38] while it also suppresses the TGF-b1 cytokine leading to apoptosis and IL-8 and IL-17 expression and inhibits the CXCR4 chemokine receptor's expression, resulting in a decreased TGF-β1-mediated CTCL cells migration [39]. Apart from verifying the apoptotic effect of bortezomib and methotrexate on CTCL, our results reveal for the first time the effectiveness of each agent and/or their combination in SS and MF cell lines. Specifically, our observations indicate that bortezomib is the most efficient agent in SS cell lines in terms of apoptosis and methotrexate in MF cells, while the two drugs do not seem to act synergistically in vitro, suggesting lack of efficacy of this combination in clinical use. We next investigated the effect of bortezomib-and methotrexate-treatment on DNA repair signaling pathways in CTCL using PCR arrays technology. Our data demonstrate for the first time that both drugs significantly alter the expression of a large number of genes involved in DNA repair systems in SS and MF cell lines, suggesting an additional possible mechanism for the enhanced apoptosis of CTCL after treatment with the aforementioned agents. These observations are further supported by an increasing number of studies which indicate a connection between aberrant DNA repair mechanisms and the pathogenesis of CTCL. Specifically, a recent study based on whole exome and SNP array based copy number analyses of CD4 + tumour cells of a large number of untreated SS patients showed various aberrations in genes related to DNA repair and genome stability, such as the POT1 and ATM genes responsible for telomere maintenance and genes associated to homologous recombination (RAD51C, BRCA2, POLD1) [40]. In addition, the aforementioned association between aberrant DNA repair mechanisms and CTCL pathogenesis has been demonstrated through the identification of a novel CTCL cell line, BKP1, derived from the peripheral blood of a SS patient. BKP1 cells bear a deletion of three genes involved in the repair of DNA damage (FANCC, FANCM and XPA), suggesting that this could lead to genomic instability [41]. Moreover, there is evidence that promoter hypermethylation in CTCL leads, among others, to deregulation of MGMT, a gene that encodes a DNA repair enzyme responsible for the removal of alkylating lesions at the guanine base protecting against mutagenesis and malignant transformation [42]. Therefore, DNA repair related deficiencies could be associated with the reported apoptotic resistance of CTCL and the related pathways should be considered as possible therapeutic targets. Most significantly, our data reveal that each agent has a different effect in terms of apoptosis' induction and DNA repair profile alterations on SS and MF cell lines. Specifically, in both SS cell lines, bortezomib induced higher apoptotic rates and presented with a greater impact on DNA repair profile than methotrexate. Interestingly, in the MF cell line MyLa, methotrexate and not bortezomib appeared with the higher impact on apoptosis while the opposite was observed in terms of DNA repair genes' expression, where bortezomib seemed to affect more genes than methotrexate. These findings could be attributed to the differences in the mechanism of action between bortezomib and methotrexate and are in accordance with previously published reports. Indeed, bortezomib has been previously associated with the regulation of DNA damage response (DDR). Specifically, the mechanisms of proteasome inhibition's lethality have been reviewed in the past and include, among others, the inhibition of the DNA repair mechanisms [43]. Moreover, the role of the proteasome in the DNA repair response has already been proposed [44,45], while proteasome inhibitors have been reported to sensitize tumour cells to DNA-damaging agents and cause a deficient DNA repair [46,47]. Howard and colleagues [48], examined the effect of bortezomib on Double strand break (DSB) repair and suggested that the influence of bortezomib on the DDR and hence on DNA crosslink sensitivity, disrupts FANC signalling and possibly blocks Alternative end joining (Alt-EJ), homologous recombination (HR), and/or end resection. There are fewer reports regarding the association of methotrexate with DNA repair systems. Nonetheless, methotrexate has been shown to affect the homologous recombination repair of DNA DSBs by controlling the expression of HR related genes and suppressing the proper assembly of HR recombination-directed subnuclear foci [49].  Our data further demonstrate that the examined SS cell lines presented similarities in their response to each agent in terms of DNA repair pathways' alterations, while the MF cell line exhibited a different profile. These observations are in line with the distinct clinical nature of SS and MF, since these diseases arise from functionally and phenotypically different cell populations, as previously reported [8,50]. Specifically, the malignant T cells in SS/L-CTCL express markers of central memory T cells (T CM ) and therefore SS is a malignancy of T CM , while those in MF express markers of skin resident effector memory T cells (T EM ), suggesting that MF is a malignancy of T EM cells [8,51]. In addition, Hut-78, SeAx and MyLa cell lines bear differences regarding their p53 status, as revealed after p53 mutation analysis. Hut-78 cells bear a homozygous nonsense mutation, SeAx a homozygous missense mutation and MyLa (MF cell line) was shown to carry wild-type p53 [52]. Therefore, in contrast to MyLa cell line, both SS cell lines lack a functional p53, which could be associated with the observed different response profile of SS and MF cell lines, given the importance of p53 in multiple cellular pathways. Indeed, p53 is a tumour-suppressor protein which responds to a wide variety of stress signals including DNA damage and ultimately decides on the cellular fate through activation of cell cycle progression, apoptosis, senescence, metabolism, and autophagy [53,54], depending on the nature of the stress signals, the type of tissue and the cellular environment [55]. The above suggest that the increased apoptotic rate of the SS cell lines after treatment with both drugs could be initiated by p53-independent pathways which compensate for p53 function, while for the MF cell line the observed apoptosis could be p53 induced, thus explaining the differential response between SS and MF. This notion is further supported by different studies showing that bortezomib is capable of inducing apoptotic cell death in both a p53-dependent and a p53-independent manner [56][57][58]. Moreover, methotrexate has been shown to induce apoptosis through a p53/p21dependent pathway [59,60]. This could further explain the reason why the wild-type p53 MyLa cell line exhibited higher apoptotic rates under methotrexate-treatment compared to bortezomib-treatment. In contrast, the p53 deficient SS cell lines respond with the higher level of apoptosis after bortezomib treatment and not methotrexate, since the proteasome inhibitor can probably initiate a p53-independent cell death program and therefore effectively induce apoptosis. Based on the above, CTCL patients, both SS and MF, could possibly benefit from a combinational treatment in a clinical level. The apoptotic rate of bortezomib in SS could be augmented by a co-treatment with agents that act in a p53 dependent manner in terms of apoptosis induction, whereas methotrexate treatment in MF could lead to an enhanced apoptotic rate when combined with an agent that acts through a p53 independent manner, apoptosiswise. However, according to our data, the combination of bortezomib and methotrexate does not manage to achieve higher death rates than monotherapy and therefore is not suggested for clinical use in either SS or MF. Further studies, initially in an in vitro level, are required in order to reveal the most successful combinations for overcoming the CTCL's resistance to chemotherapy.
In summary, we revealed that bortezomib and methotrexate induce DNA repair systems' deregulation in MF and SS suggesting another mechanism through which these agents lead to apoptosis. We also provided clinically significant information regarding CTCL therapy, showing in vitro that bortezomib is a more suitable option for the treatment of SS while methotrexate for MF. Finally, we showed that the bortezomib/methotrexate combination fails to achieve better responses compared to monotherapy in CTCL cell lines, indicating that there is no rationale for such a chemotherapeutic combination in our clinical practice.
Our observations need to be further confirmed following the results of clinical trials in CTCL patients, where the distinct nature of each condition, SS or MF, should be taken into consideration.

Author Contributions
Conceptualization: VP VM EP.