Letter Cite This: Nano Lett. XXXX, XXX, XXX-XXX
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Reprogramming Tumor-Associated Macrophages To Reverse EGFRT790M Resistance by Dual-Targeting Codelivery of Gefitinib/ Vorinostat Huige Peng,†,‡ Binfan Chen,†,‡ Wei Huang,† Yubo Tang,† Yifan Jiang,† Wenyuan Zhang,†,‡ and Yongzhuo Huang*,† †
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China University of Chinese Academy of Sciences, Beijing 100049, China
‡
S Supporting Information *
ABSTRACT: Gefitinib is a first-line therapy in the EGFR-mutated nonsmall cell lung cancer (NSCLC). However, the development of drug resistance is almost unavoidable, thus leading to an unsustainable regimen. EGFRT790M mutation is the major cause responsible for the molecular-targeting therapy failure in NSCLC. Although the recently approved osimertinib is effective for the EGFRT790M-positive NSCLC, the osimertinib-resistant EGFR mutation is rapidly developed, too. In this study, we proposed a tumor-associated macrophage (TAM) reprogramming strategy for overcoming the EGFRT790Massociated drug resistance via a dual-targeting codelivery system of gefitinib/ vorinostat that acted on both TAM with overexpression of mannose receptors and the HER-2 positive NSCLC cells. The trastuzumab-modified, mannosylated liposomal system was able to repolarize the protumor M2 phenotype to the antitumor M1 and cause the elevating ROS in the cancer cells, consequently modulating the intracellular redox balance via ROS/NOX3/MsrA axis. The suppressed MsrA facilitated the EGFRT790M degradation through 790M oxidation by ROS, thus resensitizing the EGFRT790M-positive cells to gefitinib. The dualtargeting codelivery and TAM-reprogramming strategies provided a potential method for rescuing the EGFRT790M-caused resistance to tyrosine kinase inhibitor treatment. KEYWORDS: Tumor-associated macrophages, gefitinib, vorinostat, EGFRT790M, nonsmall cell lung cancer, drug resistance drug resistance based on the “key (TKI) and lock (EGFR)” pattern because it is an unmatched race that to develop a new TKI drug costs 1−2 decades long, but a TKI-specific resistant mutation can be emerged merely within 1−2 years after application of a TKI. Therefore, it is a pressing need to seek for other strategies that can efficiently overcome EGFR mutationassociated therapeutic resistance. The important role of the tumor microenvironment (TME) in controlling tumorigenesis and progression has been well documented.8 Tumor-associated macrophages (TAM) are a major player in TME. TAM is highly plastic and can polarize to two major phenotypes: the antitumor M1 (TAM1) and the protumor M2 (TAM2). The latter is generally considered as a main “accessory to the crime” that plays an important role in promoting tumor growth.9 In many human cancers, a great population of TAM2 is associated with poor prognosis.10 There is a close association between TAM and chemoresistance, of which the mechanisms involve the release of protumor cytokines and chemokines.11 Targeting TAM has
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olecular targeted therapy has significantly changed the landscape of nonsmall cell lung cancer (NSCLC) therapy.1 Gefitinib is the first epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) marketed in the world and now has been used as a first-line treatment for metastatic NSCLC with EGFR mutations (i.e., exon 19 deletions or exon 21 L858R substitutions), setting a milestone of precision medicine in cancer therapy.2 However, approximately 50% patients develop acquired resistance to gefitinib and result in unsustainable treatment, owing to the secondary T790M mutation (EGFRT790M): substitution of threonine (T) at the 790 site with methionine (M).3 The bulky M residue creates steric hindrance against the binding of the reversible EGFR TKIs (e.g., gefitinib and erlotinib); moreover, the T790M mutation dramatically increases the ATP affinity of the L858R mutant by larger than an order of magnitude, which is believed to be the primary mechanism of T790M-associated resistance.3−5 To overcome gefitinib resistance, the third-generation TKI osimertinib (AZD9291) has been approved by the FDA for treating EGFRT790M-positive patients since 2015. However, a new drug-resistant mutation C797S has soon been found in those patients receiving osimertinib treatment.6,7 It implies the formidable challenge to overcome the EGFR mutation-related © XXXX American Chemical Society
Received: August 31, 2017 Revised: November 2, 2017
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DOI: 10.1021/acs.nanolett.7b03756 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 1. Characterization of tLGV, cytotoxicity, and apoptosis rate of H1975 cells. (A) TEM images of tLGV. (B) Cumulative release of Gef in tLGV. (C) HER-2 expression in H1975 cells and the xenografted tumor and CD206 expression in M2Φ. (D) Uptake of tLGV with or without pretreatment with trastuzumab in H1975 cells, and uptake of tLGV with or without pretreatment with mannose in M2Φ. (E) Cytotoxicity study. (F) Apoptosis rate of H1975 cells (note: drugs were only given to the H1975 chamber).
alkaline) was pH-dependent (Figure 1B), which was favorable for tumor delivery. Figure 1C shows the EGFRT790M-mutated human NSCLC H1975 cells and the transplanted H1975 tumors highly expressed HER2. The tLGV had a significantly increased uptake efficiency compared to the nontargeting LGV. When the H1975 cells were pretreated with trastuzumab, the HER2assisted uptake enhancement was inhibited (Figure 1D). It suggested the importance of the HER2-mediated endocytosis pathway. Moreover, tLGV displayed an increased uptake compared with LGV in the mouse bone marrow-derived macrophage (BMDM)-induced M2 phenotype (termed M2Φ), but no significant difference between them if the cells were pretreated with free mannose, which demonstrated the involvement of CD206-mediated endocytosis in M2Φ (Figure 1D). There was no CD206 expression found in the H1975 cells (Figure S2). Effect of TAM on Antitumor Activity. The tLGV exhibited the enhanced antitumor efficacy (Figure S1C), which could be accounted for with the synergy of the inhibition of EGFR/HDAC signaling. Importantly, the efficacy of gefitinib was enhanced when the resistant H1975 cells were exposed to the M1Φ-conditioned medium but reduced in the M2Φconditioned medium (Figure 1E). The tLGV induced higher apoptosis rate in the H1975 cells than free drugs Gef/Vor (termed GV) (19.4% vs. 26.7%, the total apoptosis cells in Q2 and Q3 areas, Figure 1F, Figure S1D). Notably, M1Φ was efficient to sensitize the H1975 cells in which the apoptosis rate was increased to 42.1% and 61.1% for GV and tLGV (Figure 1F), respectively. By contrast, there was a reduced apoptosis rate of 17.4% for GV and 23.8% for tLGV in the H1975 cells that cocultured with M2Φ (Figure 1F). TAM that constitutes up to 50% of the tumor mass is primarily M2-like, which promotes tumor progression and immunity suppression.18 The M1/M2 expression frequency correlated with extended overall survival in NSCLC patients.19 The high M1 density was positively associated with an NSCLC
been an important treatment strategy to overcome drug resistance in chemotherapy.12 For example, modulation of the polarization from TAM2 to TAM1 can resensitize the cancer cells to the cytotoxic agents and arrest the tumor growth in a drug-resistant colon cancer mouse model.13 However, there was little known about the effect of TAM on the acquired drug resistance in EGFR-TKI therapy. Specifically, the reversibility of EGFRT790M-associated resistance modulated by TAM was not demonstrated yet. We thus proposed a novel treatment strategy by reprogramming TAM polarization for reversing the EGFRT790M-assoicated gefitinib resistance. We developed a trastuzumab-modified, mannosylated liposomal system (termed tLGV) for dualtargeting the HER2 positive NSCLC cells and the mannose receptor (CD206)-overexpressed TAM2, in which gefitinib (Gef) and vorinostat (Vor) were codelivered. HER2 has been typically found with amplification in breast cancer (approximately 30%), but its overexpression in NSCLC (13−20%) has also been realized as a promising therapeutic target.14 Although trastuzumab-based treatment has been explored for HER2-positive NSCLC treatment, HER2-targeting drug delivery in lung cancer has rarely been investigated, and its feasibility has not been demonstrated yet. Vor was the first histone deacetylase inhibitors (HDACi) approved by FDA in 2006. HDACi has shown the manipulating macrophage functions for therapeutic benefits.15 For example, an HDACi TMP195 can reduce tumors and metastases via repolarization of TAM.16 Vor has been reported with the ability to inhibit the intratumor infiltration of TAMs.17 However, the effect of Vor on the TAM reprogramming was not depicted yet. Characterization and Cellular Uptake of tLGV. The trastuzumab-modified, mannosylated liposomes tLGV showed a particle size about 180 nm with a good stability (Figure 1A, Figure S1A,B). Gef and Vor are poorly water-soluble, which facilitated them in being encapsulated in the lipid membrane. The encapsulation efficiency of tLGV was 88.6% for Gef and 83.8% for Vor. In tLGV, the release profile of Gef (a weakly B
DOI: 10.1021/acs.nanolett.7b03756 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 2. MΦ phenotype polarization. (A,B) mRNA levels of the phenotype markers in M2Φ measured by qRT-PCR. (C) CD206 expression in M2Φ after drug treatment. (D) mRNA levels of the MΦ-related markers in M2Φ that cocultured with H1975. (E) tLGV upregulated the TNF-α level in the BMDM that cocultured with the cancer cells. (F) tLGV upregulated the TNF-α level in M2Φ cocultured with the cancer cells. (G) Expression of CD206, HDAC2, and TGF-β downregulated by tLGV in M2Φ and BMDM. (H) Expression of CD206, HDAC2, and TGF-β downregulated by tLGV in M2Φ and BMDM that cocultured with tumor cells.
patient’s survival time.20 The polarization regulation by the tLGV was investigated in the M2Φ. After tLGV treatment, the mRNA levels of the M2-related markers arginase I, IL-10, and CD206 in M2Φ were decreased, whereas the M1-related markers iNOS, CD86, and TNF-α were significantly upregulated (Figure 2A,B). The Western blotting results further confirmed the downregulation of CD206 by treatment with Vor, GV, or tLGV, while treatment of Gef or osimertinib yielded no change compared with the untreated control (Figure 2C). The results demonstrated the repolarization from M2Φ to M1Φ by tLGV. Furthermore, a dual-chamber transwell system was used for the M2Φ and H1975 cocultures. Similarly, tLGV also enhanced the mRNA expression of the M1-related markers, but reduced the M2-related markers (Figure 2D). The ELISA assay showed that the production of an antitumor cytokine TNF-α was significantly increased in the M2Φ cocultured with H1975 cells after tLGV treatment (Figure 2E,F). It revealed that the switch from M2Φ toward M1Φ induced by tLGV could also occurr in the presence of the cancer cells. The repolarization was further demonstrated by Western blotting. In the M2Φ or the M2Φ/H1975 cocultures treated
with tLGV, the classic M2 markers, CD206 and TGF-β, were remarkably downregulated (Figure 2G,H). It should be noted that the suppression of HDAC2 was in line with the downregulation of these M2 markers. It suggested that the inhibition of HDAC2 pathway could be involved in the M2-toM1 repolarization. Mechanisms of Overcoming EGFRT790M-Associated Resistance by TAM Repolarization. There were few reports on the effect of TAM1 on EGFRT790M. ROS-dependent oxidation of methionine residues of proteins to methionine sulfoxide is a common biochemical process in the cells; such process is readily reversed by methionine sulfoxide reductase.21 Induction of methionine 790 (790M) oxidation can cause EGFRT790M degradation, which is regulated by the redox balance between NAPDH oxidase isoform 3 (NOX3) and methionine sulfoxide reductase A (MsrA).22 MsrA protects 790M from oxidation and maintains the stability of EGFRT790M; NOX3 is stimulated by reactive oxygen species (ROS), and the NOX3 upregulation leads to the suppression of MsrA.22 TAM1 is characterized by the stimulation of ROS production. We therefore speculated that the repolarization toward TAM1 C
DOI: 10.1021/acs.nanolett.7b03756 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. Association of ROS and O2− levels with EGFRT790M resistance. (A) ROS level in H1975 cells or the coculture with M2Φ. Normalization to the M2Φ control. (B) O2− level in H1975 cells or the coculture with M2Φ. Normalization to the M2Φ control. (C) Expression of MsrA and the antiapoptosis proteins in H1975 cells or when cocultured with M2Φ. (D) EGFR and EGFRT790M gene expression in H1975 (single culture). (E) EGFR and EGFRT790M gene expression in H1975 that cocultured with M2Φ. (F) Expression of EGFR and the downstream proteins in H1975 cells or when cocultured with M2Φ. (G) Apoptotic rates in H1975 cells (note: both the macrophages and H1975 cells were treated with drugs).
reversed the EGFRT790M-associated resistance via a mechanism of the ROS/NOX3/MsrA axis. After treatment with tLGV, ROS (Figure 3A) and superoxide (O2−) (Figure 3B) levels in the H1975 cells that cocultured with M2Φ were significantly increased, compared with those in the H1975 cells alone, in line with the downregulated MsrA (Figure 3C). Both EGFRT790M and the total EGFR were remarkably downregulated (Figure 3D,E) in the H1975 cells cocultured with M2Φ. Accordingly, the total and phosphorylated EGFR and the downstream proteins (i.e., phosphorylated Akt and Erk) in the H1975 cells were inhibited (Figure 3F). The antiapoptotic proteins Bcl-2 and Bcl-xL were suppressed in the tLGV-treated H1975 cells, and the effect was more significant when cocultured with M2Φ (Figure 3C). Accordingly, after treatment with tLGV, the apoptotic rate of the H1975 cells that cocultured with M2Φ or BMDM was 51.4% or 65.5%, compared with 28.8% for the H1975 cells in the absence of M2Φ (Figure 3G, Figure S3). It demonstrated that the tLGV repolarized the “bad” M2Φ to the “good” M1Φ and thus sensitized the H1975 cells. In Vivo Imaging Studies. The tLGV exhibited the enhanced tumor-targeting efficiency compared with the LGV (Figure 4A,B), due to the overexpression of HER-2 in the
H1975 tumor. At 24 h post i.v. injection, tLGV was found to have the highest accumulation in the tumor (Figure 4C,D). Furthermore, the dual-targeting effect of tLGV was demonstrated in the tumor slices in which the colocalization of tLGV with HER2 was observed, while no obvious colocalization was found in the LGV group (Figure 4E). It was interesting that colocalization with CD206 was found, suggesting the TAM2 targeting effect of tLGV (Figure 4F). It should be noted that FcγRIV on TAM exhibited a strong binding affinity to trastuzumab,23 which could also be a contributor. In Vivo Therapy Studies. The gefitinib-resistant H1975 NSCLC mouse model was established, and the drugs were given via i.v. injection. Gefitinib only achieved minor inhibition, whereas osimertinib substantially arrested the tumor growth (Figure 5A−C). In accordance with the in vitro results, the tLGV also exhibited the remarkable suppression of tumor growth, yielding a therapeutic effect statistically comparable to osimertinib, with a tumor growth inhibition rate of 72.1% vs 68.2% (Figure 5B). The TUNEL staining showed the highest density of the apoptotic cell population in the tLGV group (Figure 5D). No obvious body weight loss and pathological changes were found, suggesting the safety of the tLGV treatment (Figure S4). D
DOI: 10.1021/acs.nanolett.7b03756 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 4. In vivo distribution and tumor-targeting delivery of tLGV. (A) In vivo imaging of LGV or tLGV. (B) In vivo radiant efficiency at the tumor sites. (C) Ex vivo imaging of the major organs. (D) Ex vivo radiant fluorescence efficiency at tumor tissues. (E) Confocal images of the colocalization of HER2 and the DID-loaded liposomes. (F) Colocalization of CD206 and the DID-loaded liposomes. Scale bars, 75 μm.
The population of TAM1 and TAM2 in the tumor tissues was determined by immunofluorescence and flow cytometry. CD80, a TAM1 marker, was greatly increased in the tLGV group in both the midterm and end of treatment (Figure 5E,F). Meanwhile, CD206, a TAM2 marker, was dramatically reduced after treatment with tLGV but maintained high expression in the groups of Gef or Osi (Figure 5E,F). The repolarization of TAM2 toward TAM1 was also indicated by FACS analysis, showing the increased TAM1 and reduced TAM2 in the tLGV group (Figure 5G,H; Figure S5). It suggested that the tLGV reeducated the “bad” TAM2 into the “good” TAM1 in the tumor microenvironment. Although osimertinib significantly arrested the H1975 tumor growth, it did not reverse the macrophage polarization as significantly as tLGV. The total macrophage population remained at a relatively stable range (e.g., 27−35% at the midterm and 22−26% at the end) in all the groups. Moreover, there was little intratumoral accumulation of the labeled MΦ that was i.v. injected to the mice (Figure S6). It indicated that the intratumor conversion could be the major mechanism but not the intratumor recruitment of macrophages from the blood. The results were in line with a previous report that Vor inhibited the intratumor infiltration of TAMs.17
Furthermore, the mechanism of the ROS/NOX3/MsrA axis was examined in the tumor tissues. The elevating level of ROS was found in the tumor dissected from the tLGV groups (Figure 5I), and accordingly, NOX3 was upregulated and MsrA suppressed (Figure 5J). The level of EGFR and its phosphorylation were also significantly downregulated by the tLGV treatment, and the downstream signaling pathways (pAkt and pErk) were suppressed (Figure 5J). Therefore, the elevation of ROS and suppression of MsrA facilitated the EGFRT790M degradation through 790M oxidation, thus resensitizing the H1975 cells to Gef. It has gradually been accepted that targeting the cancer cells solely might not be sufficient to effectively combat the malignant tumors because the cancer cells can recruit and reeducate other cells in the TME to be the “accessories to the crime”.9 Our results showed that the repolarization of TAM was effective to overcome the EGFRT790M-associated drug resistance. In addition, the repolarization of TAM2 to TAM1 resulted in the reduced anti-inflammatory cytokines (e.g., TGFβ) and the increased pro-inflammatory cytokines (e.g., TNF-α), which was also an important anticancer mechanism. E
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Figure 5. Therapeutic efficacy of tLGV in the EGFRT790M-positive H1975 tumor model. (A) Tumor growth curve. (B) Tumor weight and antitumor efficiency (compared with PBS). (C) Tumor tissue images. (D) TUNEL staining of the tumor tissue slices. Representative immunofluorescence staining tumor slices for CD80 (green, M1-associated marker) and CD206 (green, M2-associated marker) at the midterm of treatment (E) and at the end of treatment (F). (G) Percentage of MΦ in tumor tissue examined by flow cytometry at the midterm of treatment. (H) Percentage of MΦ in tumor tissue at the end of treatment. (I) ROS level (DCF dye) and liposome distribution (DID dye) in tumor tissue. (J) Expression of the related signaling proteins after treatment. Scale bars, 75 μm.
The system was able to reprogram the TAM2 toward TAM1 and trigger the ROS/MsrA/EGFRT790M transduction axis, thus providing a potential method for EGFRT790M-associated drugresistant NSCLC therapy.
In summary, we developed a trastuzumab-modified, mannosylated liposome system for dual-targeting both the TAM2 and HER2-positive NSCLC cells, thus achieving modulation of the intracellular redox balance for downregulating EGFRT790M. F
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Bronson, R. T.; Davis, S. P.; Lobera, M.; Nolan, M. A.; Letai, A. Nature 2017, 543 (7645), 428−432. (17) Tran, K.; Risingsong, R.; Royce, D. B.; Williams, C. R.; Sporn, M. B.; Pioli, P. A.; Gediya, L. K.; Njar, V. C.; Liby, K. T. Carcinogenesis 2013, 34 (1), 199−210. (18) Zheng, X.; Turkowski, K.; Mora, J.; Brune, B.; Seeger, W.; Weigert, A.; Savai, R. Oncotarget 2017, 8, 48436−48552. (19) Yuan, A.; Hsiao, Y. J.; Chen, H. Y.; Chen, H. W.; Ho, C. C.; Chen, Y. Y.; Liu, Y. C.; Hong, T. H.; Yu, S. L.; Chen, J. J.; Yang, P. C. Sci. Rep. 2015, 5, 14273. (20) Ma, J.; Liu, L.; Che, G.; Yu, N.; Dai, F.; You, Z. BMC Cancer 2010, 10, 112. (21) Stadtman, E. R.; Moskovitz, J.; Levine, R. L. Antioxid. Redox Signaling 2003, 5 (5), 577−82. (22) Leung, E. L.; Fan, X. X.; Wong, M. P.; Jiang, Z. H.; Liu, Z. Q.; Yao, X. J.; Lu, L. L.; Zhou, Y. L.; Yau, L. F.; Tin, V. P.; Liu, L. Antioxid. Redox Signaling 2016, 24 (5), 263−79. (23) Shi, Y.; Fan, X.; Deng, H.; Brezski, R. J.; Rycyzyn, M.; Jordan, R. E.; Strohl, W. R.; Zou, Q.; Zhang, N.; An, Z. J. Immunol. 2015, 194 (9), 4379−86.
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b03756. Experimental section and supplementary data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86-21-2023-1981. E-mail:
[email protected]. ORCID
Yongzhuo Huang: 0000-0001-7067-8915 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the National Basic Research Program of China (973 Program 2014CB931900, 2013CB932503), NSFC (81373357, 81422048, 81673382, 81521005), SIMM Institutional Project of CASIMM0120153023, and the CAS Scientific Equipment Development Project (YZ201437) for the support. We thank National Center for Protein Science Shanghai, CAS, for the Electron Microscopy Facility. We thank Profs. Yi Chen and Min Huang for their critical reading of the manuscript and suggestions.
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REFERENCES
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