Vani 27.11.09

Headquarters of the Australian Microscopy & Microanalysis Research Facility Australian Key Centre for Microscopy and Microanalysis ARC Centre of Excellence for Design in Light Metals

Electron Microscope Unit The University of Sydney Australia

Inhibition of histone deacetylases reduces the expression of ROCK 1 in high-density collagen matrix by indirect mechanisms Vanisri Raviraj1*, Eric Thompson2, Lilian Soon1* AKCMM, University of Sydney, Sydney, NSW, Australia, 2Department of Surgery, St Vincent’s Institute, Melbourne, VIC, Australia

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*[email protected], *[email protected]

Methodology

Introduction

Rat breast adenocarcinoma cells, MTLn3 cells were grown on high-density (20 mg/ml) 3D collagen matrix in complete AdvDMEM without antibiotics. Twenty four hour after seeding, cells were treated with different concentration of MS-275 alone or combination with other drugs for 48hr. Confocal microscopy: forty-eight hours after MS-275 treatment, cells were fixed in 4% PFA and stained in Alexa fluor 488 phalloidin. Cells were Imaged under the Olympus FV1000 confocal microscope. Live cell images were taken in GFP-actin transfected cells. Extraction of total RNA and Q-PCR: Total RNA was extracted using Trizol and RNeasy Mini extraction kit. One micro gram of total RNA was reverse transcribed using Superscript111 First strand synthesis system and 1µl of cDNA was used to quantify genes in Rotor gene 6000. Protein extraction and ROCK activity assay : HD gels were digested in 0.5mg/ml Collagenase, supplemented with 1X Kerb’s ringer buffer and 100mM CaCl2 for 30min at 37oC. Cells were pelleted at 2000rpm and washed 2 times in cold PBS. Cells were lysed in RIPA buffer and

In cancer, reduced histone acetylation is significantly correlated with advanced tumor stages and depth of the cancer invasion (1). Histone deacetylases (HDACs) have been recently shown to be important factors in cell migration and invasion for both normal and malignant cells. HDAC inhibitors are being used in clinical trails to counter cancer and they function by blocking cell proliferation, angiogenesis and cell invasion and by promoting cell differentiation and apoptosis (2). In our study, the class I HDAC inhibitor, MS-275, significantly downregulates the metastasis promoter gene ROCK1 in mRNA, protein level and activity levels. This effect is mediated by intermediate molecules (p53 and Notch).

supernatant was separated at 13,000rpm for 15min. Ten microgram of protein was used to measure activity using ROCK activity assay kit from Cell Biolabs Inc. In Brief, 10µg of protein was incubated in 65ul of 1X Kinase/ ATP/ ROCK substrate solution (MYPT1) at 37oC for 30min. Protein/substrate was separated 12% SDS gels.

Results

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1 µM MS-275

Control 0’

10’

12’

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RT-PCR for N-WASP

3 µM MS-275

2 1.8 1.6 1.4 1.2 WASP 1 0.8 Relative N0.6 0.4 0.2 0

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Inside the collagen gel

1uM MS275

Con α -tubulin

MS-275 increased the number and size of protrusions in MTLn3 cells.

RT-PCR for ROCK1 1µM MS-275

Control ROCK1

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HD(20mg/ml)

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Q u ic k T im e ªa n da d e c o m p r e s s o r a r en e e d e dtos e eth isp ic tu r e .

LD(1mg/ml)

ROCK transcript and activity levels are highly upregulated in HD matrix.

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1.8 1.6 1.4 1.2 1 Notch1 Relative 0.8 0.6 0.4 0.2 0

α -tubulin

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expression 0.2 Relative ROCK1

MS-275 down-regulates ROCK1 mRNA and kinase activity in HD matrix.

RT-PCR for Notch1

PhosphoMYPT1(T696 )

0.6 0.4

α -tubulin

3 µM

MS-275 suppress 1.2 the ROCK1 1 indirectly because 0.8 0.6 inhibition with 0.4 cycloheximide 0.2 (CHX) which blocks expression 0 Relative ROCK1 protein expression, 275 275 reversed the downCHX MS-275 Control regulation of 1uM MS-3uM MS-10ug/ml CHX+3uM ROCK1 by MS-275.

1.2

Q u i c k T i m e ª a n d a d e c o m p r e s s o r a r e n e e d e d t o s e e t h i s p i c t u r e .

3uM MS275

RT-PCR for ROCK1

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Q u ic k T im e ªa n da d e c o m p re s s o r a ren e e d e dtos e eth isp ic tu re .

Phospho -MYPT1

Relative ROCK1 Control 1uM MS- 3uM MS275 275

3µM MS-275

1µM

1uM MS275

MS-275 increased the expression of RhoB and N-WASP, genes that function in cell migration.

RT-PCR for ROCK1

1.40 1.20 1.00 0.80 0.60 0.40 0.20 expression 0.00

Control

3uM MS275

N-WASP

Live cell imaging showing protrusion- and contractility-led invasion mechanisms.

RT-PCR for RhoB

3uM MS275

RT-PCR for p53

RT-PCR for ROCK1

1.6 1.4 1.2 1 0.8 0.6 0.4 expression Relative 0.2 p53 0

Control

1.4 1.2 1 0.8 3uM 0.6 MS-275 0.4 0.2 expression 0

Relative ROCK1

1uMMS275

3uMMS275

Control 10uMDAPT 3uMMS-275 10uMPifithrin MS-275&DAPT MS-275&Pifithrin

MS-275 increased the transcripts of the tumour suppressors, Notch1 and p53, in concentration dependant manner.

Summary The micro-environment is a regulator of the expression of ROCK, which was up-regulated in HD matrix compared to LD matrix. MS-275 down-regulated ROCK1 in mRNA, protein and activity levels. Cycloheximide reversed ROCK1 suppression by MS-275 suggesting that the effects of MS-275 on ROCK1 expression is indirect. Notch1 and p53 were directly up-regulated by MS-275 and were identified as the intermediaries in controlling ROCK1 levels in cells treated with MS-275.

References 1. Tao Liu, S. K., Andrew Tee, Glenn M. Marshall (2006). "Histone deacetylase inhibitors: Multifunctional anticancer agents." Cancer treatment reviews 32: 157-165. 2. Yasui W, O. N., Ono S, Mitani Y, Ito R, Nakayama H (2003). "Histone acetylation and gastrointestinal carcinogenesis." Ann N Y Acad Sci 983.

The up-regulation of Notch1 and p53 by MS-275 is responsible for the inhibition of ROCK1; drug-ablation of p53 or Notch1 reversed the effects of MS-275 on ROCK1 levels.

Acknowledgement This study has been supported by the NHMRC (# 402510) and ARC (DP0881012). Vanisri Raviraj is funded on a NHMRC scholarship. We also gratefully acknowledge the Australian Microscopy and Microanalysis Research Facility (AMMRF), AKCMM, Sydney, Australia for staff assistance and utilization of the facility.