Tumor-Targeted Nanoparticles Deliver a Vitamin D-Based Drug

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Tumor-Targeted Nanoparticles Deliver a Vitamin D-Based Drug Payload for Treatment of EGFR Tyrosine Kinase Inhibitor-Resistant Lung Cancer Chang Liu, Tatiana Shaurova, Suzanne Shoemaker, Martin Petkovich, Pamela A Hershberger, and Yun Wu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00307 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018

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Molecular Pharmaceutics

Tumor-Targeted Nanoparticles Deliver a Vitamin D-Based Drug Payload for Treatment of EGFR Tyrosine Kinase Inhibitor-Resistant Lung Cancer Chang Liu1, Tatiana Shaurova2, Suzanne Shoemaker2, Martin Petkovich3, Pamela A. Hershberger 2,*, Yun Wu1,* 1

Department of Biomedical Engineering, University at Buffalo, The State University

of New York, Buffalo, NY, USA. 14260 2

Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer

Center, Buffalo, NY, USA. 14263 3

Department of Biomedical and Molecular Sciences, Queens University, Kingston,

Ontario, Canada. K7L 3N6

*corresponding authors Pamela A. Hershberger, Ph.D.; Department of Pharmacology and Therapeutics, MRC 232C, Roswell Park Comprehensive Cancer Center, Elm and Carlton Streets, Buffalo, NY 14263; Email: [email protected]; Phone: 716-845-1697; FAX: 716-845-8857 Yun Wu, Ph.D.; Department of Biomedical Engineering, 332 Bonner Hall, University at Buffalo, The State University of New York, Buffalo, NY 14260; Email: [email protected]; Phone: 716-645-8498; FAX: 716-645-2207

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Table of Contents


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Abstract

Mutation in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene drives the development of lung cancer. EGFR tyrosine kinase inhibitors (EGFR TKI) including erlotinib and afatinib are initially effective in treating EGFR mutant non-small cell lung cancer (NSCLC). However, drug resistance quickly develops

due

to

several

mechanisms,

including

induction

of

the

epithelial-mesenchymal transition (EMT). No effective therapies are currently available for patients who develop EMT-associated EGFR TKI resistance. 1,25-dihydroxyvitamin D3 (1,25D3) promotes epithelial differentiation and inhibits growth of NSCLC cells. 1,25D3 thus represents a promising agent for treatment of EMT-associated EGFR TKI resistance. However, 1,25D3 induces the expression of 24-hydroxylase (24OHase), which decreases 1,25D3 activity. CTA091, a potent and selective 24OHase inhibitor, has been developed to attenuate this adverse effect. CTA091 also suppresses renal 24OHase activity and so may promote hypercalcemia. To exploit favorable effects of 1,25D3 plus CTA091 in tumor cells while avoiding problematic systemic effects of 24OHase inhibition, we developed EGFR-targeted, liposomal nanoparticles (EGFR-LP) to offer tumor-targeted co-delivery of 1,25D3 and CTA091. We then established an EMT-associated model of EGFR TKI resistance, and showed that such nanoparticles improved cellular uptake of 1,25D3 and CTA091, drove pro-epithelial signaling by upregulating E-cadherin (CDH1), and significantly inhibited the growth of EGFR TKI resistant cells. Our results demonstrated that the delivery of vitamin D based drug payloads via tumor-targeted



EGFR-LP has promise as a new therapy for EFGR TKI resistant lung cancer. Future studies will focus on in vivo evaluation of biological activity, therapeutic benefits and systemic toxicity prior to clinical translation.

Keywords: vitamin D, liposomal nanoparticle, lung cancer, EGFR tyrosine kinase

inhibitor resistance


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Introduction

Approximately 24,000 individuals who have never smoked in their lifetimes die from lung cancer in the United States each year (1). A significant proportion of lung cancers in never-smokers occur in women and are driven by an activating mutation in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene (2). Mutated EGFR proteins drive lung tumorigenesis by aberrantly activating downstream pro-survival/pro-proliferative signaling pathways (3). EGFR tyrosine kinase inhibitors (EGFR TKIs) including erlotinib and afatinib are FDA-approved as first line therapy in patients with advanced EGFR mutant non-small cell lung cancer (NSCLC) and are initially effective (4-6). However, drug resistance invariably and rapidly develops (median progression free survival is 10-13 months) (7). In a majority of cases, drug resistance results from a secondary EGFR T790M mutation (8). Acquired resistance also results from EGFR bypass. Targeted therapies to overcome these two forms of resistance are either FDA-approved or in clinical trials. A third clinically relevant form of EGFR TKI resistance centers on the induction of the epithelial-mesenchymal transition (EMT) (9-13), which converts tumor cells to a variant phenotype that no longer depends upon EGFR signaling. There are currently no effective therapies for patients who develop EGFR TKI resistance due to EMT induction. 1,25-dihydroxyvitamin D3 (1,25D3) is recognized as an anti-cancer agent (14, 15). 1,25D3 elicits anti-cancer activity upon binding to the vitamin D receptor (VDR) and regulating expression of genes that control cell differentiation, apoptosis, and cell cycle progression (16, 17). The ability of 1,25D3 to support epithelial differentiation (18-21) and inhibit the growth of NSCLC cells that express VDR (including EGFR mutant NSCLC cells) is well-documented by us and others (22-24). However, 1,25D3



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activity is limited by the induction of CYP24A1 that encodes 24-hydroxylase (24OHase), the enzyme that catabolically inactivates 1,25D3. We previously discovered that the adverse effects of CYP24A1 expression on 1,25D3 activity in lung cancer cells can be overcome using the potent and selective 24OHase inhibitor, CTA091 (25, 26). CTA091 is a 24-sulfoxamine derivative of 1,25D3. It binds to the active site of 24OHase but does not bind to VDR. Unfortunately, to date, it has not been possible to exploit systemic delivery of CTA091 for cancer treatment because CTA091 also suppresses renal 24OHase activity (27). Inhibition of the renal enzyme can lead to increased blood levels of 1,25D3 that promote hypercalcemia. In order to exploit favorable effects of 1,25D3 plus CTA091 in tumor cells while avoiding problematic systemic effects of 24OHase inhibition, we hypothesize that tumor-targeted liposomal nanoparticles (LP), which co-deliver 1,25D3 and CTA091, can be used to drive vitamin D-mediated pro-epithelial signaling to restore EGFR TKI sensitivity in drug-refractory disease. LP are selected as the nanocarrier system because they have been considered so far as the most successful drug delivery system. Liposome-based drugs have been clinically used in cancer therapy (Doxil®, DaunoXome®, Depocyt®, Marqibo®, Mepact®, Myocet®, OnivydeTM), treatment of fungal diseases (Abelcet®, Ambisome®, Amphotec®), viral vaccines (Epaxal®, Inflexal® V), analgesics (DepDurTM, Exparel®) and photodynamic therapy (Visudyne®) (28). LP are a very versatile platform that not only encapsulates both hydrophilic drugs and hydrophobic drugs but also allows targeting molecules to be anchored on the surface to realize tumor-targeted drug delivery. Because the EGFR gene is amplified in >50% of EGFR mutant lung cancers (29, 30), we select EGFR as the target to preferentially deliver 1,25D3 and CTA091 to tumor. In this study, we developed EGFR-targeted LP containing both 1,25D3 and CTA091


(i.e.

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EGFR-LP-CTA091-VD).

We

characterized

the

physical

properties

of

the

nanoparticles, observed more efficient delivery by EGFR targeting, optimized the ratio of co-packaging 1,25D3 and CTA091 in EGFR-LP, and demonstrated the growth inhibitory effects of EGFR-LP-CTA091-VD in EGFR TKI-resistant NSCLC cells.

Materials and methods Materials

1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (ammonium salt) (Mal-DSPE-PEG) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol (C3045) was purchased from Sigma-Aldrich (St Louis, MO). D-α-Tocopheryl polyethyleneglycol 1,000 succinate (TPGS) was purchased from Eastman (Kingsport, TN). FAM labeled oligodeoxynucelotides (FAM-ODN) were purchased from Alpha DNA (Montreal, Canada). Human anti-EGFR/ErbB1 monoclonal antibody (MAB1095, used in nanoparticle preparation), anti-E-cadherin monoclonal antibody (Clone 36), and anti-α-tubulin antibody (Clone DM1A) were purchased from R&D systems (Minneapolis, MN), BD Transduction Labs (San Jose, CA), and Millipore (Temecula, CA) respectively. Human anti-EGFR (4405, used in western blotting) and pEGFR (Tyr1068) (2234) were from Cell Signaling Technology (Danvers, MA). Human anti-Vimentin (550513) was purchased from BD Bioscience (San Jose, CA). Traut’s Reagent (2-imiothiolane, 26101) was purchased from ThermoFisher Scientific (Grand Island, NY). Sulforhodamine B was purchased from



Sigma-Aldrich. CTA091 was kindly provided by Martin Petkovich, Queens University. 1,25-dihydroxyvitamin D3 was the generous gift of Hoffman-LaRoche (Nutley, NJ). Erlotinib was purchased from ChemiTek (Indianapolis, IN).

Cell culture and establishment of erlotinib-resistant HCC827R1 cells.

HCC827 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were expanded, authenticated by short tandem repeat profiling (IDEXX Bioresearch), and verified to be mycoplasma free. HCC827 cells were cultured in RPMI 1640 media (ThermoFisher Scientific; 11875-093) supplemented with 10% fetal bovine serum (FBS; ThermoFisher Scientific; 26140-079) and 1X penicillin streptomycin (PS; ThermoFisher Scientific; 15140-122) in 75 cm2 cell culture dishes (Greiner Bio-one; Monroe, NC). To generate an erlotinib-resistant derivative, HCC827 cells were treated with 1 µM erlotinib every 72 h for at least 30 days. Cells that survived treatment were expanded and designated as HCC827R1. Surviving HCC827R1 cells were cultured in growth medium containing 1 µM erlotinib. For maintenance, both HCC827 cells and HCC827R1 cells were seeded at the concentration of 2×105 cells/mL and incubated in a CO2 incubator at 37°C. The cells were sub-cultured every 2 days.

Sulforhodamine B (SRB) Assay

HCC827 or HCC827R1 cells were plated in 12 well plates at 3×103 cells per well in triplicate and treated with increasing concentrations of erlotinib (0-5 µM) every 72 h


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for 9 days total. Growth inhibition was determined by Sulforhodamine B (SRB) assay (31). Briefly, cells were fixed with cold 10% trichloroacetic acid for 1 h at 4°C, washed four times with tap water and dried completely. Cells were then stained with 0.057% (wt/vol) SRB in 1% (vol/vol) acetic acid for 30 min at room temperature. The dye was removed; cells were washed with 1% acetic acid four times and dried completely. Protein-bound dye was solubilized in 10mM Tris base and the OD at 510 nm was measured using a BioTek SYNERGY multimode microplate reader. The percentage of cell growth inhibition was calculated using the following formula: %ℎ ℎ = 1 −

∗ 100%

Growth curves were compared and IC50 values were determined by non-linear regression using GraphPad Prism 7.04 software.

Preparation of EGFR-LP-CTA091-VD

Lipids (DOTMA, cholesterol, DOPE, TPGS), CTA091 and 1,25D3 were dissolved in ethanol and mixed at molar ratio of DOTMA:Cholesterol:DOPE:TPGS: CTA091:1,25D3=40:36:20:1:1:2. One part of the mixture in ethanol was injected into 9 parts of HEPES buffer (20mM, pH=7.4) to produce untargeted liposome nanoparticles containing CTA091 and 1,25D3 (i.e. LP-CTA091-VD). Anti-EGFR antibody was then post inserted to LP-CTA091-VD following a previously reported protocol (32). Briefly, the anti-EGFR antibody was first conjugated with Mal-DSPE-PEG to form EGFR-DSPE-PEG. The EGFR-DSPE-PEG was then incubated with LP-CTA091-VD at the lipid to EGFR molar ratio of 2000 for 1 h at 37°C to form EGFR-LP-CTA091-VD.



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Size and zeta potential measurement

The size and zeta potential of EGFR-LP-CTA091-VD were measured by NanoBrook 90Plus Particle Size Analyzer (Brookhaven, Holtsville, NY). The samples were first diluted with deionized water. The measurements were done with temperature set at 25ºC. The data was reported as the mean diameter by volume ± standard deviation and the mean zeta potential ± standard deviation, respectively.

Encapsulation efficiency measurement

To

measure

the

encapsulation

efficiency

of

CTA091

and

1,25D3,

EGFR-LP-CTA091-VD were lysed in 1% Triton X-100 (ThermoFisher Scientific; BP151-100) to release CTA091 and 1,25D3. The absorbance at 264 nm of CTA091 and 1,25D3 was measured and the concentration was calculated based on standard curves of CTA091 and 1,25D3. The encapsulation efficiency was determined with the following equation: Encapsulation efficency (%) =

Released CTA091 and 1,25D3 × 100% Total CTA091 and 1,25D3

Cellular uptake of EGFR-LP-FAM-ODN

FAM-ODN was encapsulated in EGFR-LP to characterize the cellular uptake of nanoparticles by both flow cytometry (BD Fortessa; BD Bioscience; San Jose, CA) and laser scanning confocal microscopy (LSM 710; ZEISS; Dublin, CA). HCC827R1 cells were transfected with EGFR-LP-FAM-ODN at FAM-ODN concentration of 1 µM. Free FAM-ODN and untargeted LP-FAM-ODN were used as the controls. At 6 h post transfection, the cells were collected and fixed with 4% paraformaldehyde for the following analyses. For flow cytometry analysis, the FAM-ODN fluorescence was


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observed in the FITC channel. A total of 10,000 events were collected, and the mean fluorescence intensity of FAM-ODN was reported. For confocal microscopy, cell nuclei were stained with DAPI, and the cells were then mounted on glass slides. The fluorescence signals of DAPI and FAM-ODN were observed in the DAPI and FITC channels, respectively.

Transfection with EGFR-LP-CTA091-VD

HCC827R1 cells were seeded in 6-well plates at the density of 1.5×105 cell/well and allowed to grow overnight. Then, the cell culture medium was removed. EGFR-LP-CTA091-VD dispersed in RPMI 1640 basal media (not supplemented with erlotinib, FBS and PS) were added to cells. At 6 h post transfection, EGFR-LP-CTA091-VD were removed by replacing the RPMI 1640 basal media containing nanoparticles with 2 mL fresh RPMI 1640 media supplemented with 10% FBS, 1X PS and 1 µM erlotinib. Cells were cultured for another 48 h and then harvested for further analyses including qRT-PCR, western blotting and colony assays.

mRNA expression analysis by qRT-PCR

Total RNA was extracted from cells using TRIzol Reagent (ThermoFisher Scientific; 15596-018) followed by isopropanol precipitation. The RNA pellet was rinsed with 70% ethanol, air-dried and dissolved in DEPC water (ThermoFisher Scientific;

1504989).

The

high

capacity

cDNA reverse

transcription

kit

(ThermoFisher Scientific; 4368814) was used to reverse transcribe RNA into cDNA. Then the cDNA was amplified by real-time PCR using TaqMan gene expression assays (ThermoFisher Scientific; CYP24A1 assay ID: Hs00167999-m1, CDH1 assay



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ID: Hs01023894-m1). GAPDH (ThermoFisher Scientific; assay ID: Hs02758991-g1) was used as the endogenous control. The relative expression levels of CYP24A1 and CDH1 mRNAs in treated groups were compared to untreated controls using the ∆∆Ct method.

Protein expression analysis by western blotting

Cells were lysed using TX100/SDS buffer, as described previously (24). Proteins were quantified using the Pierce BCA Protein Assay Kit, with BSA as a standard. Equivalent amounts of total protein were resolved by SDS-PAGE and transferred to PVDF membranes for immunoblotting.

Cell viability

HCC827R1

cells

were

transfected

as

described

above

with

EGFR-LP-CTA091-VD, the mixture of free CTA091 and 1,25D3, or empty EGFR-LP. CTA091 concentration was 100nM and 1,25D3 concentration was 200nM. At 24 h post transfection, alamarBlue® cell viability reagent (ThermoFisher Scientific; DAL1025) was used to measure the cell viability following the manufacturer’s protocol. Briefly, one part of alamarBlue reagent was first diluted with 10 parts of phenol red free RPMI 1640 medium supplemented with 10% FBS and 1X PS. Then the diluted alamarBlue reagent was added to cells and incubated for 3 h at 37ºC, protected from light. Fluorescence intensity was measured by TECAN microplate reader (San Jose, CA) with the excitation and emission wavelengths at 560nm and 590nm, respectively. The cell viability of treated groups was normalized to untreated controls.


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Colony assay

HCC827R1 cells were first treated with EGFR-LP-CTA091-VD, the mixture of free CTA091 and 1,25D3, or empty EGFR-LP. CTA091 concentration was 100nM, and 1,25D3 concentration was 200nM. At 6 h post transfection, the cells were transferred to 2 mL fresh RPMI 1640 media supplemented with 10% FBS, 1X PS and 1µM erlotinib. 48 h later, HCC827R1 cells were collected, counted and then re-seeded into 6-well plates at seeding density of 1,000 cells/well. At day 3 post re-seeding, the cells were kept in the 6-well plates and transfected a second time with EGFR-LP-CTA091-VD, the mixture of free CTA091 and 1,25D3, or empty EGFR-LP. Same as the first transfection, CTA091 concentration was 100nM, and 1,25D3 concentration was 200nM. At 6 h post the second transfection, the cells were transferred to 2 mL fresh RPMI 1640 media supplemented with 10% FBS, 1X PS and 1µM erlotinib. At day 6 post re-seeding, the culture media was removed and the cells were fixed with 4% paraformaldehyde. Then 1 ml of 0.5% crystal violet (Sigma Aldrich; C0775) was added and incubated with cells for 30 min at room temperature to stain cells. Then the crystal violet was removed carefully and the cells were rinsed with tap water. The plates with colonies were left to air dry at room temperature. The numbers of colonies which had more than 50 cells/colony was determined by phase contrast microscopy.

Results

EGFR mutant lung cancer cells develop EMT-associated EGFR TKI resistance. Our prior work demonstrated that EGFR mutant lung cancer cells express VDR, vitamin D metabolites significantly inhibit the growth of erlotinib-naïve EGFR mutant lung tumor xenografts, and 1,25D3 opposes TGFβ-induced EMT in EGFR mutant



lung cancer cells (21, 24, 33). Based on these data, we hypothesized that vitamin D metabolites could also be exploited to overcome EGFR TKI resistance that results from EMT. To address this possibility, we first developed an EMT-associated model of EGFR TKI resistance. HCC827 cells (EGFR del746-750) were exposed on a chronic basis to erlotinib (1µM). Cells that survived the erlotnib treatment were expanded and designated as HCC827R1. Indicative of EMT induction, HCC827R1 cells had a more mesenchymal appearance (Figure 1a), expressed less E-cadherin, and expressed more Vimentin than parental cells at both the mRNA and protein levels (Figure 1b and 1c). EGFR was still expressed by HCC827R1 cells, suggesting that EGFR is still a valid target for tumor-targeted delivery (Figure 1c). HCC827 parental cells and HCC827R1 cells were subsequently tested for their erlotinib sensitivity in dose-response studies (Figure 1d). Cells were treated with erlotinib at concentrations ranging from 0 to 5 µM for 9 days. Dose-response curves were generated from three independent experiments per cell line. IC50 values were determined from the dose-response data using non-linear regression. The IC50 for erlotinib in HCC827 cells was 1.5 nM. The IC50 for erlotinib in HCC827R1 cells was 2.6 μM, more than 1500-fold increase compared to parental HCC827 cells.

Preparation and characterization of EGFR-LP-CTA091-VD

We previously demonstrated that the CYP24A1-encoded 24OHase restricts 1,25D3 actions in lung cancer cells, and that 1,25D3 activity could be potentiated using the 24OHase inhibitor CTA091 (25, 26). We sought to co-deliver CTA091 and 1,25D3 in a tumor-targeted nanoparticle to maximize efficacy and reduce potential systemic toxicity (Figure 2a). To generate the nanoparticles, CTA091 and 1,25D3 were mixed with lipids at CTA091 concentration of 1 mol% and 1,25D3 concentration


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of 2 mol%. The mixture was injected into HEPES buffer to quickly dilute ethanol concentration to 10% and LP-CTA091-VD were formed. Then anti-EGFR antibodies were post-inserted on the surface of nanoparticles to form EGFR-LP-CTA091-VD. This preparation method produced uniformly distributed nanoparticles with the mean diameter by volume of 157±13nm and polydispersity of 0.19 (Figure 2b). The EGFR-LP-CTA091-VD had positive surface charges with zeta potential of 13.67±2.14mV. The encapsulation efficiency of 1,25D3 and CTA091 was ~75%.

Cellular uptake

To investigate the cellular uptake of nanoparticles, fluorescent dye FAM labelled oligodeoxynucleotides, i.e. FAM-ODN, was encapsulated in EGFR-LP to form EGFR-LP-FAM-ODN. HCC827R1 cells were treated with free FAM-ODN, untargeted LP-FAM-ODN or EGFR-LP-FAM-ODN at FAM-ODN concentration of 1 µM. At 6 h post transfection, the fluorescence intensity of FAM-ODN in the cells was measured by flow cytometry (Figure 3a). Compared to untreated control, the mean fluorescence intensity of FAM-ODN was increased ~2-fold for cells treated with free FAM-ODN, ~143-fold for cells treated with LP-FAM-ODN and ~337-fold for cells treated with EGFR-LP-FAM-ODN. Confocal microscopy imaging was then used to confirm the successful accumulation of FAM-ODN, LP-FAM-ODN and EGFR-LP-FAM-ODN inside the cells, not simply attaching on the outside of cells. Confocal microscopy images further confirmed that higher FAM-ODN fluorescence intensity was observed in HCC827R1 cells transfected with EGFR-LP-FAM-ODN than those treated with free FAM-ODN and LP-FAM-ODN (Figure 3b). These results demonstrate that liposomal nanoparticles are a potent nanocarrier system that effectively delivers drugs to EGFR TKI resistant cells, and EGFR targeting further enhances the delivery efficiency.



Dose optimization of CTA091 and 1,25D3

To identify the best dosage at which to combine CTA091 and 1,25D3 to maximize potential therapeutic efficacy, HCC827R1 cells were treated with EGFR-LP containing different concentrations of CTA091 and 1,25D3. The expression of CYP24A1, the target gene of 1,25D3, was measured to determine the response to 1,25D3 exposure. The expression of E-cadherin (CDH1) was quantified to determine potential EMT reversal. To determine the optimal CTA091 dosage, HCC827R1 cells were transfected with EGFR-LP-CTA091-VD at fixed 1,25D3 concentration of 200 nM and various CTA091 concentrations (10, 25, 50, 100, 200 nM). Treatments were removed after 6 h and replaced with cell culture medium containing 1 µM erlotinib. At 48 h post transfection, compared to untreated control, more than 60-fold CYP24A1 induction was observed in HCC827R1 cells treated with EGFR-LP-CTA091-VD at CTA091 concentrations of 50nM, 100nM and 200nM (Figure 4a). The highest CDH1 expression, ~2.1-fold increase compared with untreated control, was observed in HCC827R1 cells treated with EGFR-LP-CTA091-VD at CTA091 concentrations of 100nM (Figure 4b). By taking both CYP24A1 and CDH1 expression changes into account, the concentration of 100 nM CTA091 was determined to be optimal and used for further studies. Next, to determine the optimal 1,25D3 dosage, HCC827R1 cells were transfected with EGFR-LP-CTA091-VD at fixed CTA091 concentration of 100nM and various 1,25D3 concentrations (100, 200 and 500nM). At 48 h post transfection, compared to untreated control, more than 60-fold CYP24A1 induction was observed in HCC827R1 cells treated with EGFR-LP-CTA091-VD at 1,25D3 concentrations of 200nM and 500nM. There was no significant difference in CYP24A1 expression level between


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these

two

1,25D3

concentrations

(Figure

4c).

The

cells

treated

with

EGFR-LP-CTA091-VD at 1,25D3 concentration of 200 nM and CTA091 concentration of 100 nM showed the highest CDH1 expression, ~2.1-fold increase when compared with untreated control (Figure 4d). Therefore, the concentration of 200 nM 1,25D3 was selected and used for subsequent studies.

Co-delivery of CTA091 and 1,25D3 in tumor-targeted LP improves 1,25D3 transcriptional response

To determine whether co-delivery of 1,25D3 and CTA091 in tumor-targeted LP improves 1,25D3 activity, HCC827R1 cells were treated with EGFR-LP-VD with no CTA091, EGFR-LP-CTA091-VD, or empty EGFR-LP. CTA091 was used at the concentration of 100nM and 1,25D3 was used at the concentration of 200nM. Treatments were removed after 6 h and replaced with cell culture medium containing 1 µM erlotinib. At 48h post transfection, total RNA was extracted from HCC827R1 cells and the expression of CYP24A1 and CDH1 was analyzed by qRT-PCR (Figure 5). Compared to untreated control, when cells were treated with EGFR-LP-VD with

no CTA091, the CYP24A1 expression was increased ~11-fold, but the CDH1 expression remained unchanged. When CTA091 and 1,25D3 were co-delivered via EGFR-LP, the expression levels of CYP24A1 and CDH1 were ~64-fold and ~2.1-fold higher than untreated control, respectively. No significant change in the expression of CYP24A1 and CDH1 was observed in cells treated with empty EGFR-LP. These results showed that co-delivery of CTA091 with 1,25D3 in EGFR-LP further enhanced the cellular response to 1,25D3, and thus may improve the therapeutic efficacy of 1,25D3.



Comparison of EGFR-LP-CTA091-VD versus free 1,25D3 plus CTA091

To determine whether co-delivery of CTA091 and 1,25D3 in EGFR-LP has benefit over free drug administration, HCC827R1 cells were transfected with free CTA091 and 1,25D3 mixture, EGFR-LP-CTA091-VD or empty EGFR-LP. CTA091 was used at the concentration of 100nM and 1,25D3 was used at the concentration of 200nM. Treatments were removed after 6 h and replaced with cell culture medium containing 1µM erlotinib. At 48 h post transfection, total RNA and protein of HCC827R1 cells were collected for qRT-PCR and western blotting analyses, respectively. The cells treated with EGFR-LP-CTA091-VD showed a ~64-fold increase in CYP24A1 expression and ~2.1-fold increase in CDH1 expression compared with untreated controls (Figure 6a, 6b). However, no significant difference in the expression of CYP24A1 and CDH1 was observed between untreated control, the cells transfected with the mixture of free CTA091 and 1,25D3, and the cells treated with empty EGFR-LP. At the protein level, the expression of E-cadherin was significantly upregulated in cells treated with EGFR-LP-CTA091-VD compared to untreated control (Figure 6c). These results indicate that CTA091 and 1,25D3 delivered via EGFR-LP more effectively induce a cellular response to 1,25D3. The expression of E-cadherin could potentially re-sensitize the cells to erlotinib.

Effects of EGFR-LP-CTA091-VD on HCC827R1 growth

The acute toxicity of EGFR-LP-CA091-VD was first investigated. HCC827R1 cells were treated with free CTA091 and 1,25D3 mixture, EGFR-LP-CTA091-VD or empty EGFR-LP. CTA091 was used at the concentration of 100 nM and 1,25D3 was used at the concentration of 200 nM. Treatments were removed after 6 h and replaced with cell culture medium containing 1 µM erlotinib. At 24 h post transfection,


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HCC827R1 cell viability was measured by alamarBlue assay and normalized to untreated control (Figure 7a). There was no significant difference among all treated groups and untreated control indicating that acute toxicity induced by co-delivery of CTA091 and 1,25D3 or by EGFR-LP was negligible in vitro. At 48 h post transfection, the HCC827R1 colony survival assay was initiated (Figure 7b). All groups received 1 µM erlotinib. The number of colonies that had more than 50 cells was determined after 9 days. Compared to controls, the colony formation of cells transfected with EGFR-LP-CTA091-VD was inhibited by ~30%, while the cells treated with empty EGFR-LP and free CTA091 and 1,25D3 were comparable to controls. Combining the results from the acute toxicity study and the colony survival assay, we concluded that EGFR-LP-CTA091-VD more effectively inhibited the cell growth than free CTA091 and 1,25D3, and the growth inhibition was a result of the co-delivery of CTA091 and 1,25D3, not caused by the toxicity induced by nanoparticles.

Discussion and conclusions

Approximately 20% of patients diagnosed with lung adenocarcinoma have an activating mutation in the tyrosine kinase domain of the EGFR gene (2). Patients with advanced EGFR mutant lung cancer are now treated with EGFR TKIs like erlotinib and afatinib, yielding better outcomes than cytotoxic chemotherapy (34, 35). Unfortunately, EGFR TKIs are not curative due to the rapid and invariable emergence of drug resistance. Acquired drug resistance primarily results from EGFR T790M mutation, activation of alternative or bypass pathways (such as MET overexpression), histologic transformation (which encompasses epithelial-mesenchymal transition (EMT) and transformation to small cell lung cancer (36). The third generation EGFR TKI, osimertinib, is highly effective against tumor cells harboring the EGFR T790M



mutation (37). Combination therapies are being tested to overcome resistance that results from receptor bypass. In recent phase I/II studies, the combination of MET inhibitor, tivantinib, with erlotinib significantly improved overall survival and progression free survival of NSCLC patients versus erlotinib alone (38, 39). However, no effective therapies currently exist to treat EMT-associated EGFR TKI resistance. We hypothesize that 1,25D3 may be used to reverse EMT and restore EGFR TKI sensitivity based on prior observations by us that 1,25D3 opposes TGFβ induction of the EMT in lung cancer cells (21). Beneficial effects of 1,25D3 are obtained in vitro at a concentration of 100 nM. However, 1,25D3 treatment also increases the expression of CYP24A1 and thus the levels of 24OHase, which inhibits 1,25D3 activity. Although CTA091, a potent and selective inhibitor of 24OHase, can attenuate the adverse effects of CYP24A1, it has not been applied for cancer therapy because CTA091 also suppresses renal 24OHase activity and increase the risk of hypercalcemia (27). To overcome this limitation, we sought to develop tumor-targeted liposomal nanoparticles to co-deliver 1,25D3 and CTA091 to cancer cells. We demonstrate for the first time that such nanoparticles can be used to promote epithelial gene expression (CDH1) and restore growth inhibition in EGFR TKI resistant lung cancer cells. Liposomal nanoparticles are potent and effective drug delivery systems, whose success has been demonstrated by many clinical products approved by FDA, such as Doxil®, Abelcet®, Ambisome®, Epaxal®, DepDurTM, and Visudyne®. In this study, liposomal nanoparticles have been developed to co-deliver 1,25D3 and CTA091 to maximize the therapeutic efficacy of 1,25D3 in EGFR TKI resistant lung cancer and to reduce the risk of systemic toxicity. Cationic lipid, DOTMA, is the major component of LP because it offers LP positive surface charges, facilitates the


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interaction between LP and negatively charged cell membrane, and therefore enhances the cellular uptake. DOPE and cholesterol are selected as the helper lipids to not only maintain the LP stability but also improve the drug release inside the cells (40, 41). TPGS, a PEGylated vitamin E, is used in the formulation to enhance the cellular uptake of LP and extend the circulating time in blood (42). In addition, because EGFR is overexpressed in >50% of EGFR mutant lung cancers (29, 30), and EGFR-targeted nanoparticles have shown enhanced therapeutic efficacy in cancer (43), anti-EGFR antibodies are anchored on our LP surface to achieve tumor-targeted delivery. Anti-EGFR antibodies cetuximab and panitumumab are FDA-approved, thus, cetuximab or panitumumab may be used in our nanoparticles to accelerate clinical translation. The EGFR-LP-CTA091-VD prepared by the simple ethanol dilution method showed a uniform size distribution with mean diameter by volume of 157±13 nm (Figure 2). The encapsulation efficiency of 1,25D3 and CTA091 was ~75%. Because of positive surface charge (zeta potential is 13.67±2.14 mV), in the cellular uptake study, the HCC827R1 cells treated with EGFR-LP-FAM-ODN showed ~337-fold increase in fluorescence intensity of FAM-ODN, which was significantly higher than the cells treated with free FAM-ODN (~2-fold increase). Free FAM-ODN molecules carry negative surface charges, therefore, their interaction with negatively charged cell membrane is limited by electric repulsion, leading to poor cellular uptake. Compared with cells treated with untargeted LP-FAM-ODN (~143-fold increase), EFGR targeting significantly enhanced the cellular uptake, demonstrating the effective tumor-targeted delivery (Figure 3). To investigate the therapeutic efficacy of EGFR-LP-CTA091-VD, we first optimized the ratio at which to combine CTA091 and 1,25D3. When HCC827R1 cells were treated with EGFR-LP-CTA091-VD at CTA091 concentration of 100 nM and



1,25D3 concentration of 200 nM, the expression of CYP24A1 and CDH1 was increased by ~64-fold and ~2.1-fold compared with untreated control (Figure 4). Moreover, when the HCC827R1 cells were treated with EGFR-LP-VD with no CTA091, the expression of CYP24A1 was increased by ~11-fold and the expression of CDH1 was not changed compared with untreated control, demonstrating the significant benefit of co-delivery of CTA091 and 1,25D3 (Figure 5). However, when the cells were treated with free CTA091 and 1,25D3 and empty EGFR-LP at CTA091 concentration of 100 nM and 1,25D3 concentration of 200 nM, the expression of CYP24A1 and CDH1 remained unchanged (Figure 6). It is likely that no response was observed in cells treated with free CTA091 and 1,25D3 because of the experimental design we used (i.e. cells were treated for 6 h, the treatments were removed, and the gene expression was measured at 48 h post treatment), which was selected to mimic an in vivo exposure. Due to the short-term exposure, little response was observed. However, HCC827R1 cells treated with EGFR-LP-CTA091-VD showed upregulation of CYP24A1 and CDH1 at 48 h post treatment. These results demonstrated that EGFR-LP-CTA091-VD can be effectively uptaken by HCC827R1 cells, and the CTA091 and 1,25D3 may be slowly released inside the cells for prolonged effects. The colony assay further confirmed the long-term therapeutic effects. The colony formation of HCC827R1 cells was inhibited by ~30% by EGFR-LP-CTA091-VD which compares favorably against control, free CTA091 and 1,25D3, and empty EGFR-LP (Figure 7). Importantly, the growth inhibition was not caused by acute toxicity induced by EGFR-LP (Figure 7a). In summary, we have developed tumor-targeted, liposomal nanoparticles to co-deliver 1,25D3 and CTA091 for the treatment of EMT-associated EGFR TKI resistance. Compared with free 1,25D3 and CTA091, the EGFR-LP-CTA091-VD has


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shown improved cellular uptake, significant upregulation of 1,25D3 target gene CYP24A1 and pro-epithelial CDH1, and effective inhibition of colony formation in HCC827R1 cells. Our results demonstrate that EGFR-LP may have great potential for the development of vitamin D based therapeutics for EFGR TKI resistant lung cancer. Future studies will focus on in vivo evaluation of the maximum tolerated dose, pharmacokinetics properties, biodistribution, therapeutic benefits and systemic toxicity of EGFR-LP-CTA091-VD prior to clinical translation. Although we are currently focused on the management of EGFR mutant lung cancer, we contend that the technology platform we are developing offers a potentially transformative approach to treating drug-resistant tumors. Because we are using a post-insertion method to incorporate tumor-targeting antibodies, the nanoparticles can be easily adapted to target and suppress the growth of other tumor types that retain VDR expression after EMT-mediated drug failure. For example, patients diagnosed with advanced EML4-ALK NSCLC receive crizotinib as first-line therapy (44). In EML4-ALK NSCLC cells, EMT results in crizotinib resistance (45, 46). EML4-ALK cells that are resistant to crizotinib retain VDR expression and respond to 1,25D3 (data not shown). Thus, LP-CTA091-VD (that are targeted to a cell surface marker of mesenchymal phenotype such as N-cadherin instead of EGFR) can be readily synthesized and exploited to enhance VDR activation, increase vitamin D anti-tumor activity, and overcome crizotinib failure.

Acknowledgments

Authors acknowledge the funding support from University at Buffalo, the State University of New York to Y. W. and Roswell Park Alliance Foundation, Roswell Park



Cancer Institute to P. A. H..


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Figure 1. Characterization of HCC827R1 cells. (a) Phase contrast images of parental HCC827 cells (red) and their erlotinib-resistant derivative, HCC827R1 (blue). (b) RNA was extracted from HCC827 and HCC827R1 cells for measurement of EMT markers by qRT-PCR (n=3). HCC827R1 cells underwent EMT as evidenced by loss of epithelial marker E-cadherin (CDH1) and gain of mesenchymal marker, Vimentin (VIM). Gene expression in HCC827 cells was arbitrarily assigned a value of 1.0. (c) Proteins were extracted from HCC827 or HCC827R1 following treatment with vehicle control (-) or 1µM erlotinib for 24 h. Expression of the indicated proteins was determined by western blot. (d) Erlotinib dose response studies were conducted in HCC827 or HCC827R1 cells. Cells were treated with the indicated doses of erlotinib every 72 h. The SRB assay was used to quantify growth inhibition at day 9 post-treatment. Dose-response curves were generated from three independent experiments per cell line.



Figure 2. Preparation and characterization of EGFR-LP-CTA091-VD nanoparticles. (a) Preparation of EGFR-LP-CTA091-VD nanoparticles. (b) Representative size distribution of EGFR-LP-CTA091-VD. The mean diameter by volume is 157±13nm, and the polydispersity is 0.19. (n=3 independent batches)


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Figure 3. Cellular uptake of the EGFR-LP-FAM-ODN analyzed by flow cytometry (a, b) and confocal microscopy (c). HCC827R1 cells were transfected with free FAM-ODN, untargeted LP-FAM-ODN and EGFR-LP-FAM-ODN at FAM-ODN concentration of 1 µM. At 6 h post transfection, cells treated with nanoparticles showed significantly higher fluorescence intensity of FAM-ODN compared with untreated control and cells treated with free FAM-ODN, and EGFR targeting further enhanced the delivery efficiency. (n=3, * p

Tumor-Targeted Nanoparticles Deliver a Vitamin D-Based Drug

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