Genomic co-amplification of TPX2 and AURKA with MYC cooperatively promote MYC-driven carcinogenesis

Yusuke Takahashi1,3, Paul Sheridan2 (sheridan@ims.u-tokyo.ac.jp), Atsushi Niida2, Genta Sawada1,3, Ryutaro Uchi1, Teppi Shimamura2, Hirofumi Yamamoto3, Yuichiro Doki3, Masaki Mori3, Satoru Miyano2, Koshi Mimori1

1) Department of Surgery, Kyushu University Beppu Hospital 2) Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, University of Tokyo 3) Department of Gastroenterological Surgery, Graduate School of Medicine, Suita

AcknowledgementsThe present study was supported in part by the following grants and foundations; CREST, Japan Science and Technology Agency (JST), the Funding Program for Next Generation World-Leading Research-ers (LS094), and the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research.

AbstractGenomic amplifications of chromosome 8q24 and 20q are an early and central event in tumorigenesis of various human malignant diseases. In the present study we utilize copy number and expression profiles of 148 colorectal cancer cases to demonstrate that 8q24 and 20q copy number were significantly correlated and that the activity of MYC (maps to 8q24) was dependant on the expression levels of TPX2 and AURKA (both map to 20q). In vitro assays, the aberant expression of MYC, TPX2, and AURKA generated more aggressive capabilities of anchorage independ-ent growth to normal fibroblast cells. Furthermore, knockdown of TPX2 or AUR-KA, or treatment with AURKA specific inhibitor (MLN8237) effectively suppresed cell proliferation of MYC-expressing colorectal cancer cells. These findings suggest that genomic co-amplification of 8q24 and 20q cooperatively promotes MYC-driv-en carcinogenesis. What is more, TPX2 and AURKA are ideal therapeutic targets for MYC-driven cancers owing to their both having a synthetic lethal interaction with MYC.

1. Clinical significance of MYC, TPX2, and AURKA expression in colorectal cancer

Kaplan-Meier overall survival curves of the 159 colorectal cancer patients. The overall survival of the high MYC and TPX2/AURKA expression group was significantly poorer than that of the other two groups.

Copy number correlation was analysed on array-CGH data of 148 colorectal cancer cas-es. The copy number of chromosome 8q24 and 20q was positively correlated (red ar-row).

Multiuple regression analysis

For each gene G on chromosome 20q, we performed the multiple regression

MYC_AP = β1MYC + β2XG + ε

where MYC_AP denotes the MYC activity profile, MYC denotes the MYC expression values, and XG denotes the expression val-ues of gene G.

Top ten significant chr 20q genes

Copy number correlation was also ana-lysed on public data (Tumorscape) for 161 colorectal cancer cases. The copy number of chromosome 8q24 and 20q was again posi-tively correlated (red arrow).

P-value plot for the 148 colorectal cancer clinical samples (horizontal axis) and public GEO datasets (vertical axis) from multiple regression analysis of the chromosome 20q genes. The expression levels of TPX2 and AURKA were identified as co-regulators of MYC activity.

Analysis of co-amplification of chromosome 8q24 and 20q on Tumorscape data shows positive correlation in various cancers. Num-bers in the round parentheses indicate the number of case sets of each cancer type.

Balloon plot for the 148 colorectal cancer clinical samples of MYC and TPX2 expression levels; balloon size represents MYC module activity. A similar patter was observed for AURKA.

The proposed mechanism of MYC-driven carcinogenesis in which co-ampli-fication and co-expression of TPX2 and AURKA with MYC promotes carcino-genesis.

Cell viability of colorectal cancer cells treated with AURKA inhibitor MLN8237. HCT-116 (MYC high) cells were more sensitive to MLN8237 at 50nM than DLD-1 (MYC low).

To further examine the dependence of MLN8237 on MYC activity similar assays on ectopic MYC expressing DLD-1 and mock cells were conducted. As expected, DLD-1 cells changed sensitivity to the AURKA inhibitor following up-regulation of MYC.

AURKA is known to regulate cell division by phosphorylating multiple downstream targets in mitotic aparatus and the full activation of correct mitotic localization of AURKA require its interaction with the spindle regulator TPX2. Thus, it is considered that MYC and TPX2/AURKA axis constitute an oncogenic network to promote MYC module activity and that targeting the TPX2/AURKA axis is an effective therapeutic strategy. Although a TPX2 inhibitor is not available, various AURKA inhibitors are commercially available. We applied the AURKA inhibitor MNL8237 (ali-sertib) in the present study on two colorectal cancer cell lines: HCT-116 (MYC high expression), and DLD-1 (MYC low expression). In brief it was found that MNL8237 suppressed cell proliferation more effectively and apoptotic cell death more aggressively increased in MLN8237 treated HCT-116 cells than in DLD-1 cells.

Ask me, if you’re interested, and I will try to talk you through this stuff.

MYC module activity for the colorectal cancer clinical samples was analysed using EEM. The MYC activity profile is partially explained by MYC expression (horizontal bar). We will see below that TPX2 and AURKA account for much of the variation in the MYC activity profile that is left unaccounted for by MYC expression.

Extraction of Expression Modules (EEM)

EEM is an algorithm designed to extract a set of genes, called an expression module, from expression data whose members behave coherently. EEM starts from a seed geneset that is as-sociated with some biological function. IT first tests expression coherence of the seed geneset in the expression dataset. If the coherence is statistically significant, EEM extracts a coherent subset of genes from the geneset as an expression module, and the geometric center of the corresponding expression profiles is referred to as an activity profiles of the expression module.

2. Co-amplification between chromosome 8q24 (MYC maps to) and 20q (TPX2 and AURKA map to) was observed in several cancer types

3. Heatmap of MYC module genes reveals MYC ac-tivity is not entirely explained by MYC expression

6. TPX2 and AURKA as therapeutic targets for MYC-driven colorectal cancer

5. Proliferation of colorectal cancer cells with high MYC expression was more effectively inhibited by knockdown of TPX2 and AURKA

4. TPX2 and AURKA co-regulate MYC activity

Gene Symbol P-value for β2>0CSE1L 9.59E-17TPX2 4.32E-16AURKA 5.80E-16CSTF1 7.22E-12SLMO2 5.96E-11AHCY 1.53E-10UBE2C 5.18E-10PSMA7 5.66E-09MAPRE1 6.62E-09DPM1 1.35E-08

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MYC or TPX2/AURKA: high (n=70)

MYC and TPX2/AURKA: high (n=59)

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91)

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(243)

Colorectal

(161

)

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41)

Hepato

cellul

ar (12

1)

Lung

NSC (734

)

Lung

SC (40)

Medullo

blasto

ma (12

8)

Melano

ma (11

1)

MPD (215

)

Ovaria

n (10

2)

Prostat

e (92

)

Renal

(126)

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ADNP

ADRM1

AHCY

ARFGAP1ASIPATP9A

AURKA

B4GALT5BCAS1BCAS4BCL2L1BLCAP

BMP7BPIC20orf108C20orf11C20orf111

C20orf112C20orf177

C20orf20

C20orf24

CASS4 CBFA2T2CD40 CDH22CDH4CEBPB

CHMP4B

COL9A3

CSE1L

CSTF1

CTNNBL1

CTSA

CTSZCYP24A1

DDX27DHX35

DIDO1DLGAP4

DNMT3B

DNTTIP1

DOK5

DPM1

DSN1

DYNLRB1

E2F1

EDEM2EDN3

EIF2S2

EIF6ELMO2EPB41L1

ERGIC3EYA2

FAM83D

FER1L4GDAP1L1GDF5GNAS

GSS

GTPBP5HCKHM13

HNF4AID1ITCHKCNB1

KCNG1 KCNK15KCNQ2KCNS1KIF3B

LAMA5LBP LIME1LOC388796

LSM14B

MAFB

MAPRE1

MMP9

MYBL2

MYL9MYT1

NCOA3NCOA6

NDRG3

NECAB3NFATC2

NFS1

NPEPL1OGFR

OPRL1 OSBPL2PABPC1LPARD6B

PCIF1

PCK1

PCMTD2

PFDN4

PHF20PI3

PIGTPKIGPLAGL2PLTP PMEPA1POFUT1PPDPFPPP1R16BPREX1

PROCR

PRPF6

PSMA7

PTGIS PTK6PTPN1PTPRT PXMP4RALGAPB

RALY

RBL1

RBM38RBM39

REM1

RNF114

RPS21RTEL1SALL4 SAMHD1SCAND1SDC4

SEMG2

SERINC3

SGK2SLC12A5SLC13A3SLC2A10SLC2A4RGSLC9A8SLCO4A1

SLMO2

SLPI SNAI1

SNTA1

SPAG4

SPATA2SPINLW1

SRCSS18L1

STAU1

STK4STMN3

STX16SULF2SYCP2TAF4TCEA2

TCFL5

TFAP2CTGIF2 TGM2

TH1L

TM9SF4TNNC2TOP1TOX2

TP53INP2

TP53RK

TPD52L2

TPX2

TSHZ2

TTPAL

TUBB1

UBE2C

UCKL1

UQCC

VAPBWFDC2

WISP2YTHDF1

YWHAB

ZBP1ZFP64

ZGPATZHX3ZMYND8

ZNF217

ZNF334ZNF335ZNF512B0

5

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