JNCI J Natl Cancer Inst (2018) 110(5): djx238 doi: 10.1093/jnci/djx238 First published online November 15, 2017 Article
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MAX Mutations in Endometrial Cancer: Clinicopathologic Associations and Recurrent MAX p.His28Arg Functional Characterization Christopher J. Walker*, Craig M. Rush*, Paola Dama*, Matthew J. O’Hern, Casey M. Cosgrove, Jessica L. Gillespie, Roman A. Zingarelli, Blair Smith, Maggie E. Stein, David G. Mutch, Reena Shakya, Chia-Wen Chang, Karuppaiyah Selvendiran, Jonathan W. Song, David E. Cohn, Paul J. Goodfellow Affiliations of authors: James Comprehensive Cancer Center (CJW, CMR, PD, MJO, CMC, JLG, RAZ, BS, MES, RS, KS, JWS, DEC, PJG), Department of Obstetrics and Gynecology (CJW, CMR, PD, MJO, CMC, JLG, RAZ, BS, MES, KS, DEC, PJG), Department of Chemical and Biomolecular Engineering (CWC), and Department of Mechanical and Aerospace Engineering (JWS), The Ohio State University, Columbus, OH; Siteman Cancer Center and the Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO (DGM). *Authors contributed equally to this work. Correspondence to: Paul J. Goodfellow, PhD, Obstetrics and Gynecology, 460 W. 12th Avenue, BRT 808, Columbus, OH, 43210 (e-mail:
[email protected]).
Background: Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes. Methods: MAX was sequenced in 509 endometrioid ECs, and associations between mutation status and clinicopathologic features were assessed. EC cell lines stably expressing MAXH28R were established and used for functional experiments. DNA binding was examined using electrophoretic mobility shift assays and chromatin immunoprecipitation. Transcriptional profiling was performed with microarrays. Murine flank (six to 11 mice per group) and intraperitoneal tumor models were used for in vivo studies. Vascularity of xenografts was assessed by MECA-32 immunohistochemistry. The paracrine proangiogenic nature of MAXH28R-expressing EC cells was tested using microfluidic HUVEC sprouting assays and VEGFA enzyme-linked immunosorbent assays. All statistical tests were two-sided. Results: Twenty-two of 509 tumors harbored mutations in MAX, including 12 tumors with the p.His28Arg mutation. Patients with a MAX mutation had statistically significantly reduced recurrence-free survival (hazard ratio ¼ 4.00, 95% confidence interval ¼ 1.15 to 13.91, P ¼ .03). MAXH28R increased affinity for canonical E-box sequences, and MAXH28R-expressing EC cells dramatically altered transcriptional profiles. MAXH28R-derived xenografts statistically significantly increased vascular area compared with MAXWT and empty vector tumors (P ¼ .003 and P ¼ .008, respectively). MAXH28R-expressing EC cells secreted nearly double the levels of VEGFA compared with MAXWT cells (P ¼ .03, .005, and .005 at 24, 48, and 72 hours, respectively), and conditioned media from MAXH28R cells increased sprouting when applied to HUVECs. Conclusion: These data highlight the importance of MAX mutations in EC and point to increased vascularity as one mechanism contributing to clinical aggressiveness of EC.
Received: June 19, 2017; Revised: August 16, 2017; Accepted: October 10, 2017 © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail:
[email protected].
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Abstract
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Endometrial cancer (EC) is the most common gynecologic cancer in the United States and one of a few cancer types for which both incidence and mortality are increasing (1,2). Endometrioid endometrial cancer (EEC) is the most common histologic subtype, accounting for approximately 85% of cases (3). Genetically, EEC is a highly mutated tumor type. Many driver mutations have been identified; however, our present understanding of gene defects that contribute to specific cancer processes such as invasion, metastasis, and angiogenesis is limited. Based on recent findings from The Cancer Genome Atlas (TCGA) and subsequent in silico analyses, the transcription factor MYC-associated factor X (MAX) emerged as a new genetic factor likely to influence EC tumor biology (4–6). MAX is the obligate binding partner of MYC, a master transcription factor with pro-proliferation, pro-growth, and oncogenic functions. MYC has low binding affinity for DNA, but MYC:MAX heterodimers bind E-box sequences to regulate gene expression (7,8). MAX plays an equally important role as a binding partner for members of the MAX dimerization proteins (MXDs) family including MNT, MXD1,3-4, and MGA. MAX:MXD family dimers oppose the pro-growth effects of MYC by promoting expression of differentiation and quiescence genes (9–11). Deregulation of MYC family members is seen in a variety of tumors (12). MAX abnormalities, however, are rare, with the notable exceptions of loss-of-function germline and somatic variants in pheochromocytoma and paraganglioma patients, and somatic loss-of-function mutations in small cell lung cancer (13–15). The missense MAX mutations reported by TCGA in ECs stand in sharp contrast to these loss-of-function MAX variants (4). One particular hotspot mutation discovered by TCGA, the MAX p.His28Arg mutation, has not been previously reported in primary specimens from other cancer types to date. We undertook studies to investigate MAX’s role in EC, particularly the functional consequences of the p.His28Arg mutation.
Methods Patient Materials ARTICLE
Uterine cancer samples were collected by the Division of Gynecologic Oncology, Washington University, St. Louis, from 1991 to 2010. Written informed consent for this study was obtained and approved by Washington University protocols HSC 91-0507 and HSC 93-0828 and Ohio State University protocol 2012C0116. Microsatellite instability (MSI) status and POLE mutation testing were performed previously (16–18). Nonendometrioid and POLE-mutated ECs were excluded.
Targeted Sequencing All coding exons in MAX long and short isoforms (NM002382 and NM145112, respectively) were sequenced to an average of 170 in 509 EECs using the TruSeq Custom Amplicon Kit v1.5 and a MiSeq instrument with Reagent Kit v2 (Illumina, San Diego, CA). Variants were identified using Miseq Reporter software (v2.5.1) (19,20).
Electrophoretic Mobility Shift Assays Oligonucleotide sequences used in electrophoretic mobility shift assays (EMSAs) are given in Supplementary Table 1 (available online). In vitro translated (IVT) protein or nuclear lysates were incubated at 42 C for 10 minutes, followed by incubation
with the indicated antibodies (Santa Cruz Biotechnology, Dallas, TX: c-MYC [N-262, sc-764] and MAX [C-17, sc-197]) at 25 C for 15 minutes, then addition of oligonucleotides and incubation at 25 C for 20 minutes. Gel shift assays were performed with the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, Waltham, MA). Results are representative of three experiments. Additional details are provided in the Supplementary Methods (available online).
Cell Culture 293T and AN3CA (parental and MAX-expressing) cells were cultured in DMEM (Sigma Aldrich, St. Louis, MO) with 10% fetal bovine serum (FBS). RL95-2 and Ishikawa (parental and MAXexpressing) cells were cultured in F12:DMEM (Life Technologies, Carlsbad, CA) with 10% FBS. Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial growth medium 2 (Lonza, Basel, Switzerland). Cell lines were confirmed mycoplasma negative. The estimated number of passages between authentication of cancer cell lines and completion of experiments is 25. Additional details regarding cell line origin, authentication, and forced expression of MAX are provided in the Supplementary Methods (available online).
Conditioned Media Preparation and VEGFA EnzymeLinked Immunosorbent Assay Cells were plated in duplicate in six-well plates at 1 106 cells/ well (AN3CA-derived cell lines) or 3.5 106 cells/well (Ishikawaand RL95-2-derived cell lines). After cells were attached, media was changed then collected either 24, 48, or 72 hours later. Media was centrifuged at 211 g (4 C) for 10 minutes. Supernatants were aliquoted and snap frozen on dry ice. Cells were counted to normalize VEGFA protein quantification. VEGFA levels were measured using the Human VEGF EnzymeLinked Immunosorbent Assay (ELISA) Kit (ThermoFisher Scientific). Absorbance at 450 nm was measured, and a fourparameter logistic standard curve was used to determine concentrations. Biologic duplicates were assayed in technical duplicate.
Microfluidic Sprouting Assay Microfluidic HUVEC sprouting assays were performed as described previously (21). Conditioned media from MAXWT, MAXH28R, or empty vector (EV) AN3CA cells was introduced into HUVEC-lined microchannels, and HUVEC sprouting into a central collagen channel was observed. Images of HUVEC sprouting were acquired immediately before and 24 hours after treatment with conditioned medium. Images were processed and analyzed with ImageJ software to calculate the normalized sprouting ratio for each aperture. Additional details are provided in the Supplementary Methods (available online).
In Vivo Tumor Models Six- to eight-week-old outbred, NCr-nu/nu females were utilized for flank (six to 11 mice per group) and intraperitoneal (two to three mice per group) xenograft studies. For flank xenografts, researchers blinded to tumor genotypes measured tumors twice weekly. Animal studies are covered by the Ohio State University IACUC Protocols No. A201300000141 and
C. J. Walker et al. | 519
No. mutations
A
12
Missense
6
Nonsense
0
TCGA
Frameshift
Helix-loop-helix
0 5
0
50
B
100
MAX wild-type (51 recurrences, 452 patients)
100 % survival
150 160aa
80 60 MAX mutants (6 recurrences, 21 patients)
40 20 0
HR = 4.00, 95% CI = 1.15 to 13.91, P = .03 0
No. at risk Wild-type 454
2
4 Years
6
8
381
310
215
141
Mutant 21
16
13
10
3
C Multivariable model for recurrence-free survival
5.25 (2.53 to 10.87)
< .001
Mutant MAX
2.95 (1.20 to 7.29)
.02
Advanced stage
2.01 (1.09 to 3.70)
.03
Presence of lvsi
1.86 (1.00 to 3.43)
.05
Grade 2
1.66 (0.83 to 3.29)
.15
Age > 60 y
1.23 (0.70 to 2.16)
.47
Grade 3
Figure 1. MAX mutations in endometrioid endometrial carcinoma (EEC). A) Schematic of MAX mutations identified in this study of 509 EEC samples (above) and by The Cancer Genome Atlas (below) (4), shown on the long isoform (160 amino acids). The hotspot p.His28Arg mutation seen in 12 tumors and the p.Arg60Gln mutation seen in three tumors both map to the helix-loop-helix domain (green). B) Kaplan-Meier plots show statistically significantly reduced recurrence-free survival (RFS) for women with MAX-mutant tumors. P value was determined by log-rank test. Hazard ratio and 95% confidence interval were obtained using the Mantel Haenszel approach. The survival curve was truncated at eight years. C) Multivariable analysis for RFS includes clinical variables commonly associated with outcome and MAX mutation status. P values and hazard ratios were calculated using Cox proportional hazards model. Variables included in model are those frequently shown to be prognostic in univariate analyses. Statistical significance was calculated using multivariable Cox proportional hazard tests. P values are two-sided. All statistical tests were two-sided. CI ¼ confidence interval; HR ¼ hazard ratio.
No. 2012A00000008-R1. Additional details are provided in the Supplementary Methods (available online).
Immunohistochemistry and Xenograft Vessel Quantification Immunohistochemistry was performed using a Bond Rx autostainer (Leica, Wetzlar, Germany). Automated dewaxing,
rehydration, antigen retrieval, blocking, primary antibody incubation, postprimary antibody incubation, detection, and counterstaining were performed using Bond reagents (Leica). Pan-endothelial cell antigen (MECA-32; 1:200, BD Pharmingen, 550563, San Diego, CA) with rabbit antirat IgG (1:200, Vector Laboratories, AI-4001, Burlingame, CA) and PECAM-1 (CD31; 1:1000, Santa Cruz Biotechnology, sc1506R) antibodies were used with Bond Polymer Refine Detection (Leica, S9800). Images
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P
HR (95% CI)
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were captured using the Vectra Intelligent Slide Analysis System (PerkinElmer, Waltham, MA). Quantification of vessel area was performed on MECA-32 stained slides for six random 20 images per xenograft using inform 2.1 (PerkinElmer) and imageJ. Additional information on sample preparation is provided in the Supplementary Methods (available online).
Statistical Analysis
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Associations between MAX mutation status and MSI, stage, recurrence/progression, age, lymphovascular space invasion, race, and adjuvant therapy were calculated using two-sided Fisher’s exact tests. P values for body mass index (BMI) and grade were calculated using two-sided chi-square tests. For univariate survival analysis, P value was determined by log-rank test, and hazard ratio (HR) and 95% confidence interval (CI) were obtained using the Mantel Haenszel approach. Cox proportional hazards multivariable analysis was performed using MAX mutation status and features known to be associated with outcome (18,22). The proportional hazards assumption was tested using the scaled Schoenfeld residuals and the Kaplan-Meier transformed survival times. None of the variables in the multivariable analysis was statistically significant (P> .05), indicating a lack of evidence for departure from the proportional hazards assumption. Ten patients with perioperative deaths (
