Colon cancer is estimated to be one of the most common types of cancer in Europe and the USA [1, 2]. Epidermal growth factor receptor (EGFR) is a cell surface receptor, with a vital role for cancer , regulating crucial cell functions and the expression of several extracellular matrix (ECM) molecules  which are key players in cancer, as recently reported by our research group [5, 6]. EGFR is one of the four members of the ErbB family, a subgroup of tyrosine kinase receptors. The activation of the receptor is triggered by specific ligands, mainly by EGF . EGFR has the ability to form both homodimers and heterodimers with other members of the ErbB family. The dimerization of the receptor causes cross-phosphorylation of its intracellular part and, therefore, the phosphorylated sites of the receptor act as docking sites for several signaling molecules, containing SH2 or PTB binding sites . The activation of such molecules initiates specific signaling pathways, which in turn activate several transcription factors. EGFR mainly uses four signaling pathways: Ras-Raf-Mek-Erk, PI3K-Akt, Jak-Stat, and PLCγ-PKC. Another very important and well-studied receptor of the ErbB family is also HER2/neu. Although HER2 has no natural ligands, it remains a key player into the ErbB family due to its ability to form stable heterodimers with the other receptors of the group . Concerning its signaling pathways, HER2 shares almost the same pathways with EGFR. EGFR overexpression has been associated with advanced stages of colon cancer . Due to HER2 heterodimerization with EGFR, a variety of studies in colon cancer examine the effect of the simultaneous inhibition of both receptors. More specifically, the monoclonal antibody against HER2, named trastuzumab, in combination with the specific tyrosine kinase inhibitor (TKI) against EGFR, erlotinib, suppressed growth development both in vivo and in vitro. It is noticing that the inhibition of both receptors enhanced the growth suppression as compared to that caused by pertuzumab alone . Mutations are a common phenomenon in colon cancer. K-Ras mutations are well-studied cases in colon cancer. K-Ras mutation rates in colon cancer account for approximately 20–50% [12–14] of the mutations. The mutation of K-Ras protein has resulted in the constitutive activation of the molecule, which in turn activates other downstream signaling molecules. This may be a mechanism for the resistance to EGFR-targeted therapies. For this reason, the monotherapy with anti-EGFR monoclonal antibodies is not recommended for patients with K-Ras-mutated status [15–19]. The existence of such mutations makes therapies for colon cancer, based on EGFR inhibition, much more complex. The ubiquitin-proteasome system is the main regulator of the non-lysosomatic degradation of a variety of intracellular proteins, including abnormal, misfolded, denatured, or otherwise damaged proteins. The 20S proteasome is a 700-kDa protease composed of 28 subunits arranged as a barrel-shaped structure of four heptameric rings. The two outside rings contain one copy each of seven different but related α-type subunits. Likewise, both inside rings contain one copy each of seven different but related β subunits, where the catalytic sites are located . The proteasome possesses multiple endopeptidase activities including chymotrypsin-like (β5 subunits), trypsin-like (β2 subunits) and caspase-like (β1 subunits) . Proteasome is responsible for the degradation of many intacellular proteins. Through that process, proteasome controls viable cellular functions and is strongly associated with cell survival and antiaging . Proteasome promotes cell survival mainly by regulation of specific transcription factor such as NF-kB, p53, and c-Jun etc. Its ability to promote cell viability makes proteasome a possible target for anticancer therapies. Indeed, in most cases proteasome inhibition leads to cancer cells apoptosis, both in vivo and in vitro. More specifically, in colon cancer cells, proteasome inhibition by MG-132 suppressed proliferation and inducted apoptosis. In other cases, the combined therapy with proteasome inhibitor and other chemotherapeutic agents enhanced the antitumor effects [23, 24]. Nuclear factor erythroid-derived 2 related factor 2 (Nrf2) is a key transcription factor for cell defense mechanisms that regulates the expression of electrophile and xenobiotic detoxification enzymes and drug efflux pump proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Under the basal conditions, the low constitutive amount of Nrf2 is maintained by the Kelch-like ECH-associated protein1 (Keap1)-mediated ubiquitination and proteasomal degradation system, largely localized in the cytoplasm. Interestingly, in some cases the induction of the proteasome and expression is mediated through an Nrf2-dependent manner , indicating that Nrf2 protective role may be, partly, due to proteasome expression. However, in cancer, the activation of Nrf2 may have different effects. More specifically, Nrf2 promotes cancer cell survival and development [26–28] and, therefore, enhances chemo resistance against anticancer therapies [29, 30]. EGFR is one of the proteins which are subjected to degradation by the proteasome. In several types of cancer, including colon, proteasome inhibition seems to affect the protein levels or the phosphorylation of EGFR [31-33]. Interestingly, several studies demonstrate that the simultaneous EGFR and proteasome inhibition causes enhanced antitumor effects [31, 32, 34], indicating that the combined EGFR/proteasome inhibition could be a possible therapeutic strategy against cancer. However, there are limited data regarding the involvement of EGFR on proteasome subunits' expression and activation. For this reason, in this study we evaluated whether EGF could affect, via EGFR or HER2, the gene expression and activity of the catalytic proteasome subunits in DLD-1 colon cancer cells. Moreover, we evaluated whether the activation of Nrf2 could possibly affect the proteasome. According to EGFR significance in colon cancer, we further investigated the effect of EGF on EGFR as well as cell proliferation.
MATERIALS AND METHODS
Chemicals and reagents
Dubleco's modified Eagle's medium (DMEM) fetal bovine serum (FBS), L-glutamine, sodium pyruvate, penicillin, streptomycin, gentamycin, and amphotericn B were all obtained from Biochrom AG (Berlin, Germany). The selective inhibitor CP-724,714 against HER2/neu was supplied by Selleck Chemicals. EGFR's specific ligand EGF, receptor's selective inhibitor AG1478, as well as the fluorogenic substrate SucLLVY-AMC for 20S proteasome chymotrypsin-like subunit β5 (PSMB5) and synthetic Oltipraz were purchased from Sigma Chemical Co. (St Luis, MO, USA). All other chemicals used were of the best commercially available grade.
Cell lines and culture conditions
DLD-1 (K-Ras mutant) cancer cell line was purchased from the American Type Culture Collection (ATCC) and cultured at 37°C in a humidified atmosphere of 5% (v/v) CO2 and 95% air. Cancer cells were cultured in DMEM supplemented with 10% FBS,1.0 mM sodium pyruvate, 2 mM L-glutamine, and a cocktail of antimicrobial agents containing 100 IU/mL penicillin, 100 mg/mL streptomycin,10 mg/mL gentamycin sulfate, and 2.5 mg/mL amphotericin B. All experiments were conducted in serum-free conditions.
Total RNA isolation and cDNA synthesis
Total RNA was isolated after cell lysis with guanidine thiocyanate using NucleoSpin RNA II Isolation System Macherey-Nagal (Germany). All total RNA preparations were free of DNA contamination, as assessed by RT-PCR analysis and his purification was determined by the absorbance ratios Α260/Α280. The amount of isolated RNA was quantified by measuring its absorbance at 260 nm (1 Α260unit = 40 μg/mL RNA). Total RNA was reverse transcripted with PrimeScript™ first strand cDNA synthesis kit, Takara (Takara Bio Inc., Japan).
End point and RT-PCR analysis
For end point PCR analysis, the KAPA Taq ReadyMix DNA Polymerase (KAPABIOSYSTEMS) method was used. Afterward, cDNA sequences were semi-quantitatively measured using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the “housekeeping” gene. All amplification products were separated by electrophoresis in a 1.5% agarose gel containing GelredTM Nucleic Acid Gel Stain (Biotium). Bands were visualized with a UV lamp and gels were photographed with a charge-coupled device (CCD) camera. For a semi-quantitative analysis, gene expression was determined as the relative fluorescence obtained for each molecule compared with the reference gene (GAPDH). Image analysis was performed using the program UVIpro (UVITec). RT-PCR was performed in a 20 μL mixture consisting of 10 μL KAPA SYBR FAST Master Mix (2×) Universal (KAPABIOSYSTEMS), containing Taq DNA polymerase, oligonucleotide primers (0,2 μM each), and 1 μM of template cDNA. The amplification consisted of a two-step procedure denaturation at 95°C for 3 min and 40 cycles with denaturation at 95°C for 3 sec and then annealing/elongation at 60°C for 20 sec using Rotor Gene Q (Giagen, USA). GAPDH was used as an endogenous control. All reactions were performed in triplicates and a standard curve was always included for each pair of primers for assay validation. In addition, a melting curve analysis was performed for detecting the SYBR Green-based objective amplicon. The relative abundance of mRNA of the gene of interest was deducted from the cycle number at which fluorescence increased above background level (Ct) in the exponential phase of the PCR, after normalization to the Ct of the calibrator. The sequence of primers as well as all their product characteristics and information are shown in Table 1.
|Gene||Primer Sequence||Annealing temperature (Τannealing)°C|
|End point PCR|
Determination of proteasome activity in cell monolayers
Cells were washed twice in phosphate-buffered saline and then lysated with a solution of 1 mM DTT in water for 1 h at 4°C (500 μl/well of a 6-well plate) . Lysates were centrifuged at 14,000 g for 30 min at 4°C to remove non-lysated cells, membranes, and nuclei, and protein concentrations were determined in supernatants by the method of Bradford  using bovine serum albumin as standard. Then the supernatants were assayed for the main chymotrypsin-like activity of 20S proteasome using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-AMC (Succ-LLVY-AMC; Sigma-Aldrich, Dorset, UK), as previously described . Cell lysates (50 μl) were incubated with the fluorogenic substrate (dissolved in DMSO) at final concentration 20 μM in 50 mM HEPES, 20 mM KCl, 5 mM MgCl2, 1 mM DTT pH 7.8 buffer, in a total volume of 100 μl, for 1 h at 37°C. To determine the specific degradation of Succ-LLVY-AMC by proteasome, MG-132, a proteasome inhibitor, at final concentration 200 μM, was added. The reaction was stopped by addition of 100 mM sodium acetate buffer pH 4.3 (150 μl). Fluorescence signal for AMC was monitored at 370 nm excitation and 440 nm emission in a TECAN infinite M200 (Austria) fluorometer, using free AMC as a standard. Proteasome activity was expressed as pmole AMC/μg total protein.
To determine whether these agents affect the proliferation of DLD-1 colon cancer cell line, the water-soluble tetrazolium salt (WST-1) assay was used. The assay is based on the reduction of WST-1 by viable cells, producing a soluble formazan salt. DLD-1 cells (6 × 103 per well) were plated in 100 μL cell culture medium with 10% FBS in a 96-well plate. After 24 h of plating, cells were washed twice with fresh culture medium serum free. Before the addition of EGF (20 ng/mL), cells were incubated with the specific inhibitors of EGFR (AG1478) and HER2 (CP-724,714), alone or in combination for 30 min. After pre-incubation of the inhibitors, EGF was added to the culture medium for 24 h and 48 h incubation time. Cell proliferation was determined using a microplate reader Infinite 200 (Tecan Austria Gmbtl). The absorbance was measured at a wavelength of 440 nm and a reference wavelength of 650 nm.
EGF/EGFR is the main signaling axis for the EGFR gene expression
EGFR is a vital receptor in colon cancer and a significant regulator of gene expression. Therefore, we first evaluated the impact of EGF on gene expression of EGFR. For this purpose, two specific TKIs were used: AG1478 against EGFR and CP-724,714 against HER2. Colon cancer cells after starvation in serum-free medium for 24 h were pre-incubated with the inhibitors (1 μΜ) for 30 min followed by further incubation with EGF (20 ng/mL) for 24 h. The mRNA levels of EGFR were assessed by RT-PCR analysis as described in the materials and methods section. As shown in Figure 1, EGF significantly upregulated the expression of EGFR. In addition, the inhibition of EGFR activation by AG1478 abolished the EGF induction by restoring the mRNA levels of the receptor lower to that of the control cells. On the other hand, the inhibition of HER2 activation by CP-724,714 in the presence of EGF elevated the EGFR expression. Moreover, the combined inhibition of both receptors' activity strongly decreased the levels of EGFR, reaching the levels obtained when the EGFR inhibitor AG1478 was used alone. The above data highlight that the EGF/EGFR-mediated effect of EGF is the crucial axis for the gene expression of EGFR in DLD-1 colon cancer cells.
The effect of EGF/EGFR on proliferation rate of DLD-1 colon cancer cells
It is known that EGFR regulates several cellular processes in colon cancer such as survival, differentiation, and proliferation. Taking into account that EGF affects EGFR expression, we further examined whether EGF could affect the proliferation of DLD-1 cells. As shown in Figure 2, EGF had no effect on cell proliferation. Moreover, the inhibition of the receptors alone and in combination has no regulatory effect on the proliferation of DLD-1 cell, following either 24 h or 48 h incubation periods (Figure 2). These data may well be in accordance with the existence of K-Ras mutation. However, the case that the EGF/EGFR signaling axis could affect other intracellular components crucial for cell proliferation and survival could not be excluded; therefore, we forced to evaluate the EGF effect on proteasome expression and activity.
The effect of EGF/EGFR on proteasome subunits' gene expression
Taking into consideration the above data, we then investigated the impact of EGF and EGF-mediated, EGFR, and/or HER2 inhibition on gene expression of the catalytic proteasome subunits. The proteasome subunits expression was assessed by end point PCR, as described in the materials and methods section. It is noticing that the results obtained for the expression of the three proteasome subunits exhibited a similar pattern. As shown in Figure 3, despite the presence of K-Ras mutation, EGF significantly increases the expression of β5, β1 and β2 proteasome subunits, indicating that the action of EGF overwhelms the effect of K-Ras mutation in order to induce proteasome expression. Following treatment with EGF and blocking either EGFR or both EGFR and HER2 activities, the EGF-stimulatory effects were significantly reduced. Moreover, blocking of HER2 activity alone does not suppress the EGF-enhanced expression of proteasome subunits. These data clearly show that, despite the presence of K-Ras mutation, EGF sensitizes its own receptor in order to significantly regulate gene expression of all three proteasome catalytic subunits. Interestingly, the expression patterns of proteasome and EGFR are very similar, indicating a possible correlation between them.
The effect of EGF/EGFR on β5 catalytic proteasome activity
Similar to gene expression, we evaluated the impact of EGF on the activity of the β5 catalytic proteasome subunit. For that reason, we used the same specific TKIs as described earlier, and total incubation time was 48 h. The activity of β5 (chymothrypsin-like) catalytic proteasome subunit was determined using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methylcoumarin, as described in the materials and methods section. Interestingly, the activity levels of β5 proteasome subunit (Figure 4) share a similar pattern with the expression levels of the subunit (Figure 3A). Particularly, EGF significantly increases the β5 proteasome subunit activity levels, despite the presence of K-Ras mutation. Moreover, after stimulation with EGF, the inhibition of HER2 activity does not change the EGF-mediated effect, whereas the blocking of EGFR activity or of both EGFR/HER2 abolished the stimulatory effect of EGF. According to the results, it is suggested that EGF significantly regulates the activity of the β5 proteasome subunit via the EGFR, without HER2 involvement.
Activation of Nrf2 does not affect the gene expression of proteasome subunits
Since it was known that EGF induces the activation of Nrf2 , and in turn the activation of Nrf2 induces, in some cases, proteasome expression , we examined the impact of the activation of Nrf2 on gene expression of the catalytic proteasome subunits in DLD-1 colon cancer cell lines. For this experiment, the Oltipraz, a synthetic activator of Nrf2, was used. Colon cancer cells were incubated with Oltipraz (30 μΜ) for 24 h and then the proteasome subunits gene expression was determined by end point PCR analysis, as described in the materials and methods section. As shown in Figure 5, the Oltipraz did not exhibit any significant effect on gene expression of all three catalytic proteasome subunits, indicating that in DLD-1 colon cancer cells the EGF-mediated-induced proteasome expression is not depended on the Nrf2 activation.
Several in vivo and in vitro studies relate the importance of EGFR in colon cancer development and progression. Through its pathways, EGFR is able to regulate vital cellular processes as proliferation, survival, and metastasis. Moreover, EGFR is responsible for the expression of several molecules which contribute to cancer promotion. The inhibition of EGFR results in antitumor effects both in vivo and in vitro. A very common co-receptor for EGFR is HER2. In colon cancer, the combined EGFR/HER2 activity inhibition enhances the anticancer effect, indicating interplay between those two receptors. Moreover, K-Ras is a very important signaling molecule downstream of EGFR and HER2, which is often mutated in colon cancer. The existence of K-Ras mutations induces carcinogenesis and demonstrates resistance against anti-EGFR therapies. The ubiquitin-proteasome system is the main regulator of the non-lysosomatic degradation of a variety of intracellular proteins. Several studies highlight the significance of the proteasome in several types of cancer since proteasome inhibition induces apoptosis of cancer cells. Proteasome affects the protein levels of EGFR and its phosporylation, in several types of cancer including colon [39–42]. This indicates a correlation between EGFR and proteasome. In one study, proteasome inhibition affected some signaling molecules downstream of EGFR, which in turn affected Nrf2 activation, also implicating the transcription factor in the correlation of EGFR and proteasome . Thus, it has been demonstrated that proteasome is able to modulate EGFR; however, it is not known whether the reverse option is possible. Taking into consideration previous studies showing that EGF appears to regulate the survival time of Caenorhabditis elegans by activating proteasome  and that EGF increases proteasome expression through activation of EGFR in prostate cancer cells , our basic goal was to evaluate the effect of EGF/EGFR and then HER2 on the expression and the activity of the catalytic proteasome subunits. It is worth mentioning that in DLD-1 cancer cells, the introduction of exogenous EGF leads to phosphorylation of EGFR, as previously demonstrated . As shown in Figure 1, EGF also significantly upregulated the expression of EGFR. As previously reported, EGF upregulates the expression of EGFR in breast cancer MDA-MB-231 cells through NF-κB activation . The phosphorylated EGFR activates NF-κB through PI3-K pathway leading to the phosphorylation of IκBα kinase on serine-32 and -36; therefore, promoting the nuclear translocation of NF-κB in PC-3 prostate cancer cells . This pathway may also be responsible for the observed upregulated expression of EGFR by its ligand EGF. Moreover, it is known that IκBα kinase phosphorylates the NF-κB inhibitor IκBα, which subsequently is ubiquitinated and degraded by proteasome, leading in the release of transcription factor and its nuclear translocation. Given that EGF/EGFR signaling axis strongly enhances both the proteasome expression and activity overwhelming the action of KRas mutation (Figures 3 and 4), it may be plausible to suggest that an auto-regulatory mechanism of EGF/EGFR signaling exists in DLD-1 cancer cells. In this regard, it might be that EGF activates EGFR, which in turn enhances the expression and activation of proteasome that then contributes to the activation of NF-κB pathway which finally mediates the upregulation of EGFR gene expression. It has been reported that the activation of Nrf2 is able to induce proteasome expression [49–51]. Although the involvement of Nrf2 in basal expression of human proteasome genes was hinted by the correlation of increased proteasome gene expression with overexpression of Nrf2 in human colon cancer cells , the synthetic activator of Nrf2, Oltipraz, did not exhibit any significant effect on proteasome subunits expression (Figure 5). It may be attributed, however, to K-Ras mutation. As previously proposed, the phosphorylation of Nrf2 by several kinases including, PKC, MAPK, and PI3K/Akt may affect the activation of this transcription factor and subsequently the regulation of Antioxidant Response Elements (ARE)-responsive genes. These upstream kinases for Nrf2 phosphorylation are the downstream of EGFR pathway and, in particular, downstream of activated Ras in EGFR pathway [53, 54]. Consequently, in K-Ras-mutated DLD-1 cells, it is expected Nrf2 to be already activated and thus the Oltipraz does not exhibit any effect. However, the observed enhancement of proteasome expression and activity by EGF may suggest that an additional or exclusive pathway is implicated in this process. As previously reported , EGF increases proteasome expression, almost exclusively through JAK-STAT3 signaling pathway in prostate cancer cells, while the MAPK and PI3K signaling pathways do not actually get involved in that process. This could also be a possible scenario in DLD-1 cells and could partly explain the fact that despite the presence of K-Ras mutation, EGF significantly regulates proteasome expression and activity. Several reports have suggested that Nrf2 mediates feedback induction of proteasome genes in mammalian cells when the proteasome activity is compromised [55, 56], and also it plays an important role in upregulating proteasome genes in response to oxidative stress [51, 57]. Nrf2 is not the sole transcription factor responsible for basal expression of human proteasome genes. It has been demonstrated that the CCAAT box-binding transcription factor NF-Y binds promoters of a large set of human proteasome genes that carries the CCAAT box . Some proteasome genes may be regulated by other transcription factors in addition to Nrf2 and NF-Y. It was suggested that the heat shock factor 2 (HSF2) may play a role in regulating basal transcription of mouse proteasome genes . It is known that both enhanced proteasome and EGFR activation contribute to increased tumor survival, while proteasome and EGFR inhibitions result in apoptosis in several types of cancer including colon [10, 23, 24]. Therefore, it is plausible to suggest that EGF enhances cancer cells survival through the activation of proteasome, whereas the inhibition of EGFR may partly cause apoptosis due to proteasome inhibition. This may explain that the combined EGFR/proteasome inhibition results in enhanced antitumor effect [31, 32, 34]. In addition, the proteasome may regulate the EGF/EGFR pathway by contribution in degradation of EGFR by lysosomes, as previously reported . According to the significance of EGFR in colon cancer, we evaluated the action of EGF on the expression of its own receptor and the proliferation of DLD-1 cells. Our data suggest that EGF/EGFR signaling axis is the main mediator for the expression of the receptor. Interestingly, the expression patterns of proteasome and EGFR were very similar. On the other hand, although EGFR is a vital receptor for colon cancer cells, the proliferation rates of DLD-1 cells were not affected by EGF/EGFR system, possibly due to existence of K-Ras mutation. In summary, we report that EGF/EGFR signaling axis significantly regulates the expression and the activity of the catalytic proteasome subunits in DLD-1 colon cancer cells, overwhelming the action of K-Ras mutation. Moreover, EGF/EGFR signaling axis regulates the EGFR expression in a similar mode that regulates proteasome, indicating an EGFR/proteasome correlation. The conclusions are summarized in Figure 6. These data provide the basis for more studies, which will declare the pathway(s) that EGF enhances EGFR expression and activated EGFR regulates proteasome expression, and how this could contribute to the development of more effective anticancer approaches targeting on EGFR and proteasome in colon cancer.