Academic year: 2017-2018 Somadri Ghosh A signalling function of phosphatidylinositol 3,4-bisphosphate in cell migration of breast cancer cells

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Université libre de Bruxelles

IRIBHM

A signalling function of

phosphatidylinositol 3,4-bisphosphate in

cell migration of breast cancer cells

Somadri Ghosh

Academic year: 2017-2018

Promotor: Prof. Christophe Erneux, PhD

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Composition of the jury

President of the jury: Prof. Philippe Lebrun

External expert: Prof. Christophe AMPE

External expert: Prof. Jean-Baptiste Demoulin

Member: Prof. Nicolas BAEYENS

Member: Prof. Jean-Pierre BRION

Member: Prof. Pierre Roger

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Acknowledgements

Firstly, I would like to express my sincere gratitude to my advisor Prof. Christophe Erneux for providing the continuous support of my Ph.D study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis.

I would like to thank the members of doctoral committee Prof Carine Truyens and Dr. Xavier De Deken for their insightful comments and encouragement. I would also like to thank Prof. Jean-Marie Vanderwinden for his support and suggestions throughout the thesis in regards to microscopic imaging and image analysis. I would like to offer my special thanks to Prof. Sabine Costagliola for giving me the opportunity to work in the microscope from her lab for live imaging. I would like to express my great appreciation to Dr. Isabelle Pirson and Dr. Benjamin Beck for their useful critiques on this research work. My grateful thanks are also extended to Dr. V. De Maertelaer for her help in doing the statistical data analysis, and all my collaborators who believed in me for the work.

I am particularly grateful to Televie and the Rose Foundation & Jean Hoguet funding to financially support the completion of my PhD thesis.

I would also like to extend my thanks to Ms. C Moreau, the technician of the laboratory for her kind help with various experiments during the project. I would like to thank my fellow lab mates with special mention of Dr. Elong Edimo, Dr. Anna Raquel Ramos, Dr. Sandra Koenig and Mr.Mathieu Antoine for their constant support during the research and for all the nice time spend over the last five years. Last but not the least, this PhD would not have been completed without the constant support provided by all the staffs and members of IRIBHM. My sincere thanks goes to Dr. Rashna Bhandari to introduce me to the field of phosphoinositols and phosphoinositides. I would also extend my thanks to all my friends for their support and understanding during the period.

I would also like to thank my parents, my wife and my family for their constant support and understanding especially during the hard times to pursue my dream of completing this Ph.D. thesis and life in general.

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2. Phosphoinositides (PIs) ... 14 2.1 PI monophosphates ...16 2.1.1 PI(3)P ... 16 2.1.2 PI(4)P ... 18 2.1.3 PI(5)P ... 19 2.2 PI bisphosphates ...20 2.2.1 PI(4,5)P2 ... 20 2.2.2 PI(3,4)P2 ... 22 2.2.3 PI(3,5)P2 ... 23 2.3 PI trisphosphates ...24 PI(3,4,5)P3 ... 24 3. PI phosphatases: ... 25 3.1 PI 3-phosphatases: ...25

3.1.1 PTEN and PTEN related proteins: ... 26

3.1.2 MTM and MTM related proteins: ... 27

3.2 PI 4-phosphatases: ...28

3.2.1 INPP4A/B: ... 28

3.3 PI 5-phosphatases: ...29

3.3.1 Synaptojanin 1 and Synaptojanin 2... 30

3.3.2 OCRL-1... 31 3.3.3 INPP5B ... 32 3.3.4 INPP5J ... 32 3.3.5 SKIP ... 33 3.3.6 SHIP1/2 ... 33 3.3.7 Pharbin ... 34 4. SHIP2 ... 34

4.1 Structure and enzymatic activity: ...34

4.2 SHIP2 and insulin signalling ...36

4.3 SHIP2 interaction and cytoskeletal network ...36

4.4 SHIP2 inhibitors: ...39

4.5 SHIP2 and Disease:...40

4.5.1 SHIP2 in opsysmodysplasia:... 40

4.5.2 SHIP2 and breast cancer: ... 40

5. Phosphoinositides in cell migration and adhesion: ... 44

II. Aims of the thesis ... 47

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III. Results and Methods ... 51

Chapter I: SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells. ... 52

Chapter II: Fibroblasts derived from patients with opsismodysplasia display SHIP2-specific cell migration and adhesion defects. ... 77

Chapter III: Inhibition of SHIP2 activity inhibits cell migration, induces apoptosis and prevents metastasis in MDA-MB-231 breast cancer cells. ... 88

IV. Discussion and perspectives ... 140

1. Can the control of cell migration in 1321 N1 cells be generalized to other glioblastoma cells? ... 141

2. How does SHIP2 regulate cell motility in breast cancer cells? ... 142

3. Can we generalize the regulatory role of SHIP2 to non-cancerous cells? ... 144

4. Can we use AS1949490, a commercially available SHIP2 inhibitor, as a SHIP2 probe in cell migration? ... 145

5. SHIP2 and cell adhesion, how are they related? ... 146

6. Does SHIP2 affect the survival of breast cancer cells? ... 147

7. A new pathway to control PI(3,4)P2 via PTEN ... 148

8. Does SHIP2 play a role in tumour progression and metastasis of breast cancer cells? . 149 References ... 151

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Summary

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SHIP2 is a phosphatase that belongs to the family of the phosphoinositide 5-phosphatases. It is known to dephosphorylate PI(3,4,5)P3 to PI(3,4)P2 imparting a tight control of the PI 3-kinase pathway. Over the last decade, SHIP2 has been described as a tumor promotor or tumor suppressor in several cancer types such as glioblastoma, colorectal cancer or breast cancer cells. Several studies have proposed a role of SHIP2 in breast cancer cells, but its tumor promoting function was unclear at the beginning of this thesis especially in terms of its mode of regulation. In 2013, the INPPL1 gene that encodes SHIP2 has been found to be mutated in opsismodysplasia (OPS), a rare autosomal recessive disease characterized by delayed bone maturation but no molecular mechanism was provided to explain the mechanism.

In this thesis, we first contributed to establish a negative regulation of SHIP2 on cell migration in 1321 N1 glioblastoma (GBM) cells. Our studies revealed a dephosphorylation activity of SHIP2 on PI(4,5)P2 at the plasma membrane to control cell migration. This study was done in collaboration with Dr. Elong Edimo in the lab. We have also shown that the regulation of cell motility cannot be generalized to all the GBM cells. In LN229 and U-251 GBM cells we observed a positive regulation of cell migration by SHIP2.

We next took advantage of a unique model comparing fibroblasts derived from non-affected and OPS patients (in collaboration with Dr. Valérie Cormier-Daire). We have shown that the fibroblasts from the OPS patients are SHIP2 deficient and migrate slower as compared to fibroblasts from non-affected individuals.

Finally, the major part of the thesis was the study of breast cancer cells: in the model MDA-MB-231 cells, we established a positive regulation of SHIP2 on cell migration. We extended this regulation on cell migration to different breast cancer cell models using a SHIP2 inhibitor AS1949490. We confirmed that this inhibitor blocks the phosphatase activity of SHIP2 and showed its selectivity towards SHIP2 in cell migration assay. In MDA-MB-231 cells we deciphered a second messenger role of PI(3,4)P2 to control cell migration. Our data in this model rely on the use of SHIP2 depleted cells obtained by lentiviral infection and shRNA. We confirmed the positive role of SHIP2 on cell migration in the model of rat chondrosarcoma SHIP2CRISPR cells (in collaboration with Dr. Pavel Krejci).

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A major goal of this thesis was achieved thanks to in-vivo studies: using MDA-MB-231 cells injected in SCID mice, we found a tumor promoting role of SHIP2 by determining the tumor weight. We also observed less lung metastasis of SHIP2 depleted injected cells as compared to control cells suggesting SHIP2 to be important for invasiveness of triple negative breast cancers.

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Abbreviations

ARF ADP-ribosylation factor

ARP2/3 Actin related protein 2/3

BrdU 5-bromo-2’-deoxyuridine

BSA Bovine serum albumin

Btk Burton tyrosine kinase

C-terminal Carboxy terminal

DAG Diacylglycerol

ECM Extracellular matrix

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

ER Endoplasmic reticulum

Erk Extracellular signal-regulated kinase

ESRCT Endosomal sorting complexes required for transport

FACS Fluorescence activated cell sorting

FAK Focal adhesion kinase

FCS Fetal calf serum

FN Fibronectin

GAPDH Glyseraldehyde-3-phosphate dehydrogenase

GBM Glioblastoma

GFP Green fluorescent protein

GOLPH3 Golgi phosphoprotein 3

HER2/ErbB2 EGF-like growth factor receptor

IHC Immunohistochemistry

INPP4A/B Inositol polyphosphate 4-phosphatase A/B

MAP Mitogen activated protein

MEF Mouse embryonic fibroblasts

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MTM/MTMR Myotubularin/ Myotubularin related

mTOR Mammalian target of rapamycin

NHS Normal horse serum

N-terminal Amino terminal

N-WASP neuronal Wiskott-Aldrich Syndrome Protein

OCRL Occuloceribrorenal lowe syndrome

OPS Opsysmodysplasia

PDK1 3-phosphoinositide dependent protein kinase-1

PH Pleckstrin homology

PI Phosphoinositide

PI(3)P Phosphatidylinositol 3-phosphate

PI(3,4)P2 Phosphatidylinositol 3,4-bisphosphate

PI(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate

PI3K PI 3-kinase

PKB/Akt Protein kinase B

PLC Phospholipase C

PR Progesterone receptor

PtdIns PhosphatydilInositol

PTEN Phosphatase and tensin homologue deleted on chromosome 10

SAM Sterile alpha motif

SH2 Src homology 2

SHIP SH2 domain containing inositol 5-phosphatase

SNX Sorting nexin

SYNJ1/2 Synaptojanin ½

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TAPP Tandem-PH-domain containing protein

TGN Trans golgi network

WAVE WASP-family verprolin-homologous protein

WT Wild type