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Abstract

Tyrosine kinase inhibitors (TKIs), a novel group of target-specific anti lung cancer drugs, have recently been found to resistant to some NSCLC cells which have the T790M EGFR mutation. However, recent investigations on the therapies of resistance to EGFR-TKIs are very limited. Therefore, it is important to develop more effective therapies to reverse EGFR-TKIs resistance. In our present study, erlotinib was used as the TKIs drug and the effects of the erlotinib on cell growth were evaluated. Cell viability and concentration dependent studies were performed using HCI-H1975 and HCI-H1299 cells alone with erlotinib, respectively. Further combined with rituximab, the results showed that erlotinib and rituximab were significantly inhibited the cell growth. Furthermore, the combination of erlotinib and rituximab greatly decreased the expression of p-mTOR and p-EGFR. Additional results from western blotting and immunofluorescence assays demonstrated that the accumulation of rictor was also decreased on MAM. Thus, all these results suggested that EGFR-TKIs combined with CD20 mono-antibody significantly decrease the cell growth of H1975 cells and H1299, with T790M EGFR mutation, and inhibit the localization of the key mTOR pathway proteins to MAM. So, it may be a promising strategy for overcoming EGFR TKI resistance in NSCLC patients.

Keywords

Rituximab tyrosine kinase inhibitors ER-membrane NSCLC cells

1. Introduction

EGFR is a receptor tyrosine kinases (RTKs), facilitate tumorigenic abnormal signaling, which is highly expressed in NSCLC [1, 2]. Several tyrosine kinase inhibitor (TKI) therapies against EGFR is currently administered and is initially effective in L858R mutations NSCLC patients [3, 4, 5, 6, 7]. However, the overall efficacies of TKIs are limited due to the development of drug resistance [8, 9, 10]. T790M mutations may cause primary EGFR TKI resistance. However, attributed to the development of the T790M mutations, greater than 50% of NSCLC patients may acquire TKI resistance [2]. Therefore, it is important to elucidate the mechanisms of EGFR TKI resistance in order to reverse drug resistance and develop more effective therapies.

CD20 is an important differentiation antigen of B cells. It is firstly expressed in the pro/pre B cells. Most evidence indicates that CD20 is an important molecule that regulates the signal transduction and tumor drug resistance [11, 12, 13]. According to a series of biological response to produce CD20 and anti CD20 antibody binding, we indicated that CD20 may be involved in cell proliferation, differentiation, signal transduction and calcium ion transfer [14, 15, 16]. Since the intracellular region of CD20 is a non covalent binding with tyrosine protein kinase and serine/threonine protein kinase, CD20 and anti CD20 binding will stimulate the expression of these kinases such as EGFR [17]. However, the physiological role of CD20 has not yet been clarified.

Furthermore, mammalian target of rapamycin (mTOR) is a serine/threonine kinase, which plays a key role in the PI3K/Akt pathway [18]. mTOR kinase acts in two functionally different multi-protein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2), whose activities are regulated by complex co-factors. Recent discoveries suggest a distinct role for mTORC2 in cancer [9, 20]. Originally, mTORC1 regulates many cell growth factors to integrate mitogen to control cell proliferation. By contrast, mTORC2 has been shown to function as an important regulator of the cytoskeleton, which controls the phosphorylation of Akt through the interaction between rapamycin-insensitive companion of mTOR (rictor) [21, 22, 23]. Localization of mTORC2 in the cell has been suggested to be at the plasma membrane [24] But the function of mTORC2 at MAM is not clear.

This study demonstrates that combining rituximab with current EGFR-TKIs erlotinib may successfully inhibit cell proliferation in EGFR T790M-positive mutations NSCLC cells. Furthermore, in the case of T790M-positive cells, the localization of rictor is changed by combining rituximab with EGFR TKIs. This study suggests that the mechanism of rituximab to overcome resistance to EGFR inhibitor is altrering the location of rictor from the MAM. This indicates that selective combinatorial treatment should be used for cells with T790M-positive mutated EGFR, to improve NSCLC patient prognosis.

2. Materials and methods

2.1 Reagents and antibodies

Erlotinib [N-(3-ethynylphenyl)-6,7-bis(2-methoxy-ethoxy) quinazolin-4-amine] (Cat. No. E-4007) was purchased from LC Laboratories (Woburn, MA), rituximab [3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano [4,3-d]pyrimidin-4-one] (Cat. No. 53113) was purchased from Sigma-Aldrich (St. Louis, MO). The two inhibitors were suspended in DMSO and stored as aliquots at $-$ 20 ${}^{\circ}$ C. Phosphospecific rabbit monoclonal antibodies for p-mTOR (Ser 2448, Clone D9C2), p-4E-BP1 (Thr37/46, Clone 2855), p-GSK3 $\beta$ (Ser 9), Total GSK3 $\beta$ , Axin1 (C76H11) and p-LRP6 (C5C7), phosphospecific rabbit polyclonal antibodies for p-ERK1/2 (Thr202/Tyr204) and p-p70S6K (T389), rabbit and mouse IgG secondary antibodies were obtained from Cell Signaling Technology. Rabbit polyclonal unphosphorylated GATA-6 (sc-9055) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal antibody for active $\beta$ -catenin (05665) was obtained from Millipore (Billerica, MA). A mouse monoclonal antibody for $\beta$ -actin was obtained from Sigma-Aldrich (St. Louis, MO). All antibodies were used according to the manufacturer’s instructions.

2.2 Cell lines and cell culture

NCI-H1975 and NCI-H1299 cell lines were purchased from American Type Culture Collection (ATCC) (Rockville, MD, USA, CRL-5908 and CRL-5803, respectively). The NCI-H1752 and NCI-H1299 cells expressed both the L858R and T790M EGFR mutations (documented by ATCC). The two cell lines were all stored in incubators at 37 ${}^{\circ}$ C with 5% CO ${}_{2}$ and were cultured in Roswell Park Memorial Institute (RPMI 1640) media (Thermo Fisher Scientific, Pittsburg, PA, Cat No: SH3002701) supplemented with 10% (v/v) Fetal Bovine Serum (Biological Industries, Israel, Cat No: 04-001-1A).

2.3 MTT cell viability assay

The effects of various TKIs on H1975 and H1299 cell viability were measured by MTT assay using MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide dye according to the manufacturer’s instructions. Cells were seeded at 5000 cells per well on 96-well plates and after 24 hours, cells were treated with inhibitors for 72 hours. After added the MTT reagent (Sigma-Aldrich, MO, Cat. No. M5655), cells were incubated at 37 ${}^{\circ}$ C for 4 hours. Finally, the formazan crystals were dissolved and the absorbance was measured at a wavelength of 490 nm by an automatic ELISA analyzer. All cell viability experiments were performed three times in replicates of eight for each treatment condition.

2.4 mTORC1 and mTORC C2 activities

Immunoprecipitations (IPs) of $\sim$ 200 $\mu$ g of crude protein extract of cells treated with different drugs, commenced with an overnight incubation at 4 ${}^{\circ}$ C using 2–3 $\mu$ g of primary antibodies (e.g., anti-total mTOR or anti-Rictor). Protein A/G-Agarose was added for an additional 3 h. IPs were harvested at 2000 $\times$ g for 1 min at 4 ${}^{\circ}$ C and washed two times in TBS. For kinase reactions, the samples were washed two more times with kinase assay buffer. To determine mTORC1 activity, we used the specific mTORC1 substrate, p70S6K-GST fusion protein (KLISA™mTOR Activity (Calbiochem), according to the suggested protocol). To detected mTORC2 activity, we used the mTORC2 substrate, inactive recombinant Akt-GST fusion (K-LISA™ Akt Activity (Calbiochem). Each assay is ELISA-based in a total reaction volume of 100 $\mu$ l. Substrate phosphorylations were detected using respective phospho-specific antibodies and the absorbance was recorded at 450 nm, against a reference 595 nm.

2.5 Isolation of subcellular fractionation

Cells were washed twice in PBS and pelleted by centrifugation, resuspended and lysed using a motor-driven Potter-Elvehjem homogenizer. The supernatant from pelleted homogenates was then centrifuged at 10,000 $\times$ g to separate microsomal (microsomes and cytosol) from crude mitochondrial (MAM and mitochondria) fraction. The pellet of the mitochondria centrifugation was used as the purified mitochondria in our analysis. All of the fractions were resuspended in sucrose homogenization medium and analyzed by Western blot analysis.

2.6 Immunofluorescence

Thirty thousand cells were plated in cover glass slides in 12-well (Corning Glass Works, Corning, NY) per well. Cells were allowed to adhere for 24 hours. Cells were then treated with Erlotinib/rituximab. After fixing using 4% paraformaldehyde solution in PBS, cells were then permeabilized in 0.1% Triton X-100 solution and blocked in buffer containing 5% normal goat serum. Cells were incubated with Rictor primary antibody (1:400) at 4 ${}^{\circ}$ C overnight and then incubated with IgG DyLight 488 anti-mouse secondary antibody (1:250) (Thermo Fisher Scientific, Rockford, IL) for 2 hour at room temperature. The cells were also stained with, Rhodamine Phalloidin/FITC-Phalloidin (Invitrogen, Carlsbad, CA), Mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) and 4’,6-diamidino-2-phenylindole (DAPI, Burlingame, CA). Cells were observed using a Zeiss LSM 510 META confocal microscope. Average nuclear fluorescence intensity of Rictor staining was measured using NIH ImageJ software over 10 microscopic fields per treatment condition and values were averaged.

2.7 Immunoblotting

Following the drug-treatments, cells were lysed in RIPA buffer (20 mM Tris, 150 mM NaCl, 10% sodium deoxycholate, 1% NP-40, 1 nM sodium orthovanadate, 10 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride, (Sigma-Aldrich). Cell lysates were then electrophoresed for separation on 10% SDS-PAGE. Separated proteins were then transfer to PVDF membrane for Western blot detection (Bio-Rad Laboatories, Hercules, CA) and incubated with specific EGDR or mTOR pathway related proteins antibodies overnight at 4 ${}^{\circ}$ C, then incubated with horseradish peroxidase-labeled antibody IgG (ZSGB-BIO, Beijing, China) for 1 h at room temperature. Immunoblots signals were detected by using Pierce ECL Substrate chemiluminescence kit (Thermo Fisher Scientific, Rockford, IL, 32109), and the data were calculated by Odyssey infrared imaging system using Image J software.

2.8 Statistical analyses

Data were expressed as mean $\pm$ standard error. Differences were tested by two-tailed $t$ -test. The values $P<$ 0.05 were considered statistically significant. Statistical analysis was done using GraphPad Prism software ( $t$ -test).

Figure 1.

Rituximab induces sensitivity to erlotinib in NSCLC cells harboring T790M mutation EGFR. (A) IC ${}_{50}$ for erlotinib alone or combine erlotinib with rituximab in H1975 and H1299 cells. (B) Western blotting detected the expression of PARP. (C) Synergistic inhibition of H1975 and H1299 cells was observed when erlotinib and rituximab were administered in combination with different concentration (2 $\mu$ M or 6 $\mu$ M). ANOVA analysis was used to determine differences between treatment conditions which were statistically significant ( $n=$ 3, ${}^{*}p<$ 0.05).

3. Results

3.1 Comparison of IC ${}_{50}$ of NCI-H1975 cell lines and NCI-H1299 cell lines with NCI-H2170

It was confirmed that NCI-H1975 and NCI-H1299 cells were the T790M EGFR mutation cells. MTT cell viability assays were performed for determining the IC ${}_{50}$ of H1975 and H1299 cells upon drug treatments. We used NCI-H2170 cells as control in this study, which are wild-type EGFR cells. The IC ${}_{50}$ were calculated using Sigma Plot 10 software (Sigma Plot 10.0, Systat Software Inc, IL), and the results are showed in Table 1. Compared to H2170 cells, the IC ${}_{50}$ of erlotinib in H1975 was found to be 12-fold greater, and the IC ${}_{50}$ of H1299 cells was found to be 10-fold higher. The result showed that H1975 and H1299 cells are naturally resistant to erlotinib, due to the presence of the T790 mutation.

Table 1
IC50 of Erlotinib for NSCLC cell lines with and without T790M

Cell lline	Erlotinib
NCI-H1975	8.92 $\mu$ M
NCI-H1299	7.68 $\mu$ M
NCI-H2170	0.74 $\mu$ M

Figure 2.

Modulation of key mTOR proteins in TKI-resistant cells H1975 and H1299. (A) Western blot analysis expression of p-mTOR and p-EGFR after erlotinib or erlotinib combined with rituximab treatment; (B) After western blot analysis increased expression of EGFR and mTOR pathway related proteins in H2170-ER and H2170-SR cells were observed when compared to H2170-P cells with the exception of p-GSK-3Î, in H2170 SR cells. The fold changes were calculated using ImageJ software ( $n=$ 3, ${}^{*}p<$ 0.05). (C) The mTORC1 and mTORC2 activity assay in; (D) The localization of LC3 was detected to evaluate the autophagic pathway after erlotinib or erlotinib combined with rituximab treatment. DNA was counterstained by using DAPI (bule). Images were taken with laser scanning confocal microscope (Zeiss) with a 63Ã— objective lens, scale bar, 10 $\mu$ m.

3.2 EGFR-TKI combined with CD20 monoclonal antibody inhibits the proliferation on erlotinib resistant cells

Since the T790M EGFR mutation is known to confer erlotinib resistance (Table 1) in NSCLC, it is important to find ways to reverse drug resistance caused by this mutation. It was suggested that CD20 may stimulate the exression of these kinases such as EGFR. Therefore, we tested for drug synergism on H1975 cells using rituximab, which is CD20 monoclonal antibody, via MTT cell viability assay. As shown in Fig. 1A, cell viability of both H1975 and H1299 cells were modestly inhibited by erlotinib and rituximab at lower concentrations but only erlotinib group. We confirmed the result by detecting the level of apoptosis marker PARP (Fig. 1B). Furthermore, we found that the synergistic effects on H1975 and H1299 cells growth inhibition were observed with erlotinib and rituximab in combination at concentrations of 2 $\mu$ M (erlotinib) and 6 $\mu$ M (rituximab) (Fig. 1C). However, their combinatorial effects were not synergistic above the concentration of 10 $\mu$ M of each drug (data not shown).

Figure 3.

Rituximab significantly restrains the localization of the component mTORC2. (A) mTORC2 component rictor is present in the ER fraction of cells. Total, whole cell lysate; cyto, cytoplasmic extract; ER, heavy membrane fraction from isopycnic flotation. Equal total protein levels were loaded in each lane. (B) Immunofluorescent staining with monoclonal anti- rictor antibody (green) in H1975 cells. ER/mito was stained by ER-RFP plasmid, F-actin was stained by using rhodamine-conjugated phalloidin (purplish red). DNA was counterstained by using DAPI (bule). Images were taken with laser scanning confocal microscope (Zeiss) with a 63 $\times$ objective lens, scale bar, 10 $\mu$ m. (C) Quantification of the fluorescent intensity was performed by using Image J ( $n=$ 30, ${}^{*}p<$ 0.05).

3.3 Low concentrations of Erlotinib combined with rituximab decreased the phosphorylation state of mTOR

In order to elucidate mechanism of sensitivity to erlotinib combined with rituximab in TKI-resistance cells, expression levels of key proteins involved in mTOR/EGFR pathway were determined by immunob-lotting. We observed that active p-EGFR was downregulated 4-fold in the presence of erlotinib and rituximab respectively, in H1975 cells, when compared to only erlotinib group in H1975 cells. Furthermore, we observed that p-mTOR was also downregulated 3.0-fold in the presence erlotinib and rituximab in H1975 cells when compared to erlotinib treatment in H1975 cells. Similarly, in the H1299 cells, we observed p-EGFR was downregulated 4.0-fold in the presence erlotinib with rituximab; p-mTOR was downregulated 2.5 fold in the presence erlotinib with rituximab, when compared to a single agent of erlotinib treatments in H1299 cells ( $p<$ 0.01) (Fig. 2A). No significant modulation in the expression of total proteins was observed among the differently groups (Fig. 2B). We also detected the activity of mTORC1 and mTORC2 in lysates from H1975 cells with Erlotinib or Erlotinib combined with rituximab. In lysates from such cells that were treated with Erlotinib and rituximab, the mTORC1 activity is significantly reduced compared with only Erlotinib treatment group. On the other hand, we also found mTORC2 (Rictor) activity significantly reduced after combined Erlotinib with rituximab (Fig. 2C).

To investigate whether the treatment with Erlotinib and rituximab induced the autophagy, we performed immunofluorescent staining sassay to detected the localization of LC3. We found that combined Erlotinib with rituximab could induce LC3 accumulation with the punctate structures characteristic of autophagy (Fig. 2D). These results indicated that Erlotinib with rituximab promote the recruitment and accumulation of LC3 and may be involved in the autophagy process.

3.4 Rituximab increased the sensitive to EGFR-TKI resistance cells by inhibiting mTORC2 localize to MAM

Previous studies have suggested that mTORC2 localize at the ER (39, 40), and the localization of mTORC2 to MAM is regulated by growth factors. So, we examined the localization of mTORC2 in the treatment of erlotinib with rituximab. First, we observed lower levels of mTORC2 (rictor) at MAM in H1975 cells upon erlotinib with rituximab stimulation compare with single erlotinib treatment (Fig. 3A). Second, we observed a significant decrease in colocalization of rictor with ER/mitochondria in H1975 and H1299 cells, grown in the presence of erlotinib and rituximab, compared with the cells upon erlotinib treatment only by immunofluorescence (Fig. 3B and 3C). These results suggested that the localization of mTORC2 to MAM is inhibited by the stimulation combined erlotinib and rituximab.

4. Discussion

To the best of our knowledge, the study was the first to discuss the combination treatment with EGFR-TKI and CD20 mono-antibodies to NSLCLs. However, studies have shown that EGFR phosphorylation was not inhibited by erlotinib in T790M EGFR mutation cells, accompanied by modestly promotion of cell proliferation [25, 26, 27]. So the lung cancer cells with T790M EGFR mutation were not highly sensitive to EGFR-TKIs. Indeed, the first generation of anti-EGFR therapies were all directed against the wild-type receptor. Based on the results of our study, lung cancers with T790M mutant EGFR are not sensitive to EGFR-TKI drugs, in contrast to wild-type EGFR cells. Thus, combining EGFR-TKI with the other drugs, which can reverse the resistance, might be a better therapeutic strategy for lung cancer. There is a voluminous literature that documents the successful use of CD20 monoclonal antibodies in the immunotherapy of cancer. Recent studies suggested that CD20 may stimulate the expression of these kinases such as EGFR [17]. However, the role of CD20 in the context of the EGFR-TKI setting remains unknown. In the present study, we determined the effect of the combination erlotinib with rituximab on the cell viability. The results suggest their combinatorial effects offer a promising strategy for EGFR-TKI resistance.

In our study, we measured the expression of the key proteins of mTOR during the drugs treatment. To identify the regulation of these proteins on EGFR-TKI resistance, we detected the expression of p-EGFR, p-mTOR and p-Rictor in EGFR-TKIs resistant cells H1975 and H1299 cells. There was a statistically significant down-regulation of p-EGFR and p-mTOR in the group which combine erlotinib with rituximab, compared to only erlotinib treatment ( $p<$ 0.05). Mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) is a sub-compartment of the ER, which controls mitochondrial physiology and apoptosis [28]. The main function of this membrane is to facilitate the transfer of lipids and calcium between the two organelles. MAM also mediates ER homeostasis and lipid biosynthesis by harboring chaperones and several key lipid synthesis enzymes [29]. Many recently research suggested that mTORC2 has been physically and functionally linked to both the ER and mitochondria. During erlotinib with rituximab treatment, we also observed that the level of rictor is decreased at mitochondria-associated ER-membrane (MAM). Our results suggest that the mTOR signaling pathway might play a role in the response to EGFR-TKI resistance. Treating NSCLCs with rituximab would enrich mTORC2 at MAM, with the over-expression and over-activation of mTORC2 consequently triggering the activation of EGFR, thus activating downstream signal transduction molecules, dependent of EGFR kinase activity, and finally overcoming EGFR-TKI resistance.

In summary, we demonstrate that cell viability of lung cancer cells HCI-H1975 and NCI-H1299 with T790M EGFR mutation, treated with erlotinib and rituximab in combination at lower concentrations, which is not up to 10 $\mu$ M, was significantly inhibited compared with single drug of erlotinib or rituximab. The combined action of the two drugs affects NSCLC cells survival. The localization of mTORC2 (rictor) is changed by combing rituximab to erlotinib, which from MAM to cytoplasm. Although the role of the localization of mTORC2 in disease remains to be clarified, it is tempting to speculate that novel therapeutic of combining multiple drugs could be beneficial effects of NSCLC cells which resistance to EGFR-TKI drugs. Because overcoming resistance to EGFR-TKI is now undergoing clinical evaluation; therefore, Our study might predict the resistance to EGFR-TKI and allow for individualized treatment.

Footnotes

Acknowledgments

This study was supported by Grands of Shanghai Council for Science and Technology (No. 15DZ19301 03).

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Rituximab effectively reverses Tyrosine kinase inhibitors (TKIs) resistance through inhibiting the accumulation of rictor on mitochondria-associated ER-membrane (MAM)