Immunotherapy Targeting Folate Receptor Induces Cell Death Associated with Autophagy in Ovarian Cancer Yunfei Wen1, Whitney S
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Published OnlineFirst November 21, 2014; DOI: 10.1158/1078-0432.CCR-14-1578 Cancer Therapy: Preclinical Clinical Cancer Research Immunotherapy Targeting Folate Receptor Induces Cell Death Associated with Autophagy in Ovarian Cancer Yunfei Wen1, Whitney S. Graybill2, Rebecca A. Previs1,WeiHu1, Cristina Ivan3, Lingegowda S. Mangala1, Behrouz Zand1, Alpa M. Nick1, Nicholas B. Jennings1, Heather J. Dalton1, Vasudha Sehgal4, Prahlad Ram4, Ju-Seog Lee5, Pablo E. Vivas-Mejia6, Robert L. Coleman1, and Anil K. Sood1,3,7 Abstract Purpose: Cancer cells are highly dependent on folate metab- the low-FRa A2780 model. MORAB-003 reduced prolifera- olism, making them susceptible to drugs that inhibit folate tion,buthadnosignificant effect on apoptosis. Protein receptor activities. Targeting overexpressed folate receptor alpha expression and cDNA microarray analyses showed that (FRa) in cancer cells offers a therapeutic opportunity. We inves- MORAB-003 regulated an array of autophagy-related genes. tigated the functional mechanisms of MORAB-003 (farletuzu- It also significantly increased expression of LC3 isoform II and mab), a humanized mAb against FRa, in ovarian cancer models. enriched autophagic vacuolization. Blocking autophagy with Experimental Design: We first examined FRa expression in an hydroxychloroquine or bafilomycin A1 reversed the growth array of human ovarian cancer cell lines and then assessed the in inhibition induced by MORAB-003. In addition, alteration vivo effect of MORAB-003 on tumor growth and progression in of FOLR1 gene copy number significantly correlated with several orthotopic mouse models of ovarian cancer derived from shorter disease-free survival in patients with ovarian serous these cell lines. Molecular mechanisms of tumor cell death induced cancer. by MORAB-003 were investigated by cDNA and protein expression Conclusions: MORAB-003 displays prominent antitumor profiling analysis. Mechanistic studies were performed to deter- activity in ovarian cancer models expressing FRa at high levels. mine the role of autophagy in MORAB-003–induced cell death. Blockade of folate receptor by MORAB-003 induced sustained Results: MORAB-003 significantly decreased tumor growth autophagy and suppressed cell proliferation. Clin Cancer Res; 21(2); in the high-FRa IGROV1 and SKOV3ip1 models but not in 1–12. Ó2014 AACR. Introduction metastatic foci and recurrent tumors (4). Studies that have exam- ined the functional significance of FRa in ovarian cancer cells have The folate receptor 1 gene (FOLR1) encodes a 38-kDa GPI- shown that higher receptor expression is associated with greater anchored protein (folate receptor alpha, or FRa) that binds folic biologic aggressiveness (5). acid with high affinity and transports folate by receptor-mediated Previous studies showed that anti-FRa antibodies such as LK26, endocytosis (1). FRa is selectively overexpressed in approximately Mov18, and Mov19 have reached development stages and been 90% of nonmucinous ovarian carcinomas, but is largely absent tested for clinical relevance (6, 7). MORAB-003 (farletuzumab), from normal tissues (2, 3). Its expression is maintained on which is a humanized anti-FRa mAb derived from optimization of the LK26 molecule; it maintains an affinity similar to the original 1Department of Gynecologic Oncology and Reproductive Medicine, murine LK26 antibody (approximately 2 nmol/L) and a tissue- The University of Texas MD Anderson Cancer Center, Houston, Texas. binding profile consistent with FRa distribution (5, 8). It has been 2 Department of Gynecologic Oncology, Medical University of South shown to inhibit cell growth and activate antibody-dependent, cell- Carolina, Charleston, South Carolina. 3Department of Systems Biolo- gy, The University of Texas MD Anderson Cancer Center, Houston, mediated cytotoxicity (5, 8, 9). Although MORAB-003 has been Texas. 4Department of Systems Biology, The University of Texas MD shown to act through antibody-dependent cellular cytotoxicity in 5 Anderson Cancer Center, Houston, Texas. Center for RNA Interfer- vitro (10), additional mechanisms underlying its function are likely. ence and Non-Coding RNA, The University of Texas MD Anderson Cancer Center, Houston, Texas. 6Department of Experimental Thera- Herein, we reported that sustained treatment with MORAB-003 peutics,The University of Texas MD Anderson Cancer Center, Houston, antibody in xenograft models of human ovarian cancers or in three- Texas. 7Department of Cancer Biology, The University of Texas MD dimensional (3D)–cultured ovarian cancer models expressing high Anderson Cancer Center, Houston, Texas. levels of FRa induced enrichment of late-stage autophagic events, Note: Supplementary data for this article are available at Clinical Cancer eventually leading to cell death and tumor growth inhibition. Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Anil K. Sood, The University of Texas MD Anderson Cancer Center, 1155 Herman Pressler Drive, Houston, TX 77230. Phone: 713-745- Materials and Methods 5266; Fax: 713-792-7586; E-mail: [email protected] Cell lines and cultures doi: 10.1158/1078-0432.CCR-14-1578 Human ovarian cancer cells (A2780, HeyA8, SKOV3ip1, and Ó2014 American Association for Cancer Research. IGROV1) were cultured in RPMI-1640 cell culture medium www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 4, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst November 21, 2014; DOI: 10.1158/1078-0432.CCR-14-1578 Wen et al. vised by The University of Texas MD Anderson Cancer Center Translational Relevance Institutional Animal Care and Use Committee. Development and In this study, we discovered that targeting folate receptor characterization of the orthotopic mouse models of advanced alpha (FRa) in ovarian cancer cells with specific humanized ovarian cancer have been described previously (11, 17). antibody MORAB-003 (farletuzumab) resulted in prominent antitumor activity. Our studies revealed that MORAB-003 In vivo MORAB-003 therapeutic dose finding inhibited tumor growth via induction of autophagy-associated To determine the optimal dose and schedule of MORAB-003 cell death. This inhibitory effect of MORAB-003 can be reca- therapy, 5 mice were injected i.p. with SKOV3ip1 cells to induce pitulated in three-dimensional in vitro models. Blocking tumors. One week later, treatment was initiated with either autophagy reversed the growth inhibition induced by control IgG or MORAB-003 (5, 25, or 50 mg/kg) twice or thrice MORAB-003. Moreover, data from The Cancer Genome Atlas weekly for 28 days. Mean tumor weights were recorded after 4 revealed that copy-number gains of the FOLR1 gene signifi- weeks of therapy for each treatment group. cantly correlated with shorter disease-free survival in patients with the immunoreactive subtype of ovarian serous carcino- In vivo murine ovarian cancer models ma. Taken together, our results revealed a novel functional Mice were injected with tumor cells [1 Â 106 cells/mL (SKO- mechanism of MORAB-003 in inhibiting ovarian cancer V3ip1 and A2780) or 2 Â 106 cells/mL (IGROV1) in Hank growth. balanced salt solution] to induce tumors; 1 week later, tumor- bearing mice were randomized into four groups (n ¼ 10 mice/ group for SKOV3ip1 or IGROV1 models, and n ¼ 5 for A2780 model or control) and treated with i.p. injections of the following agents: (i) control IgG (5.0 mg/kg twice weekly), (ii) MORAB-003 supplemented with 15% FBS and 0.5% gentamicin (11, 12). All (5.0 mg/kg twice weekly), (iii) control IgG (5.0 mg/kg twice cell lines were purchased from the ATCC and authenticated by the weekly) plus docetaxel (35 mg/mouse weekly), or (iv) MORAB- Cell Line Core Facility at The University of Texas MD Anderson 003 (5.0 mg/kg twice weekly) plus docetaxel (35 mg/mouse Cancer Center and routinely tested to confirm the absence of weekly). Mice were monitored daily for adverse effects and killed Mycoplasma. if they became moribund. After 4 to 5 weeks of therapy, all of the For the 3D models, cells were grown on nonadhesive optical mice were killed and necropsies were performed. For each mouse, plates (MatTek Corporation) and then brought to the glass body and tumor weight, tumor distribution, number of tumor surface, which was precoated with 200 mLofgrowthfactor– nodules, and amount of ascites were recorded at necropsy. Tissue reduced Matrigel (BD Biosciences). Approximately 18,000 to specimens were fixed in formalin for paraffin embedding or snap 20,000 spheroid-like cells were plated in each well of a 24-well frozen in optimal cutting medium (Miles, Inc.) for frozen slide plate and incubated at 37 C for 1 hour to allow cells to seed preparation. gradually. Spheroids were cultured with full medium contain- ing 2% Matrigel (13). The culture conditions and growth of Tandem mRFP/mGFP-LC3 fluorescence microscopy 3D spheroids were monitored daily in preparation for drug pGFP–RFP–LC3 (Addgene) plasmids (14) or EGFP–LC3 plas- treatment. mids (16) were stably transfected into epithelial ovarian cancer cells (HeyA8 and SKOV3). After 3D culture on glass-bottom Reagents and transfection optical 35-mm2 dishes (MatTek Corporation), expression of The pEGFP–RFP–LC3 plasmid in lentiviral vector was derived EGFP and RFP was visualized with a laser scanning multiphoton from Addgene plasmid 21074 (Addgene; ref. 14). The PEA-15 confocal microscope (TCS SP5 MP; Leica). siRNA (ggaagacauccccagcgaatt) was described previously (15). The EGFP-LC3 plasmid was derived from Addgene 11546 (16). Apoptosis assay The