Functional analysis of proteomic biomarkers and targeting glioblastoma stem cells

Radovan Komel1, Ivana Jovčevska1, Neja Zupanec1, Mirjana Liovič1, Filip Mihalič1,

Daniela Cesselli2 , Clara Limbaeck Stokin3, Serge Muyldermans4, Uroš Smrdel5, Andrej Vranič6, Boštjan Matos6

1Medical Centre for Molecular Biology Faculty of Medicine, University of Ljubljana, Slovenia

2Azienda Ospedaliero-Universitaria di Udine (AOUD),

S. Maria Della Misericordia, Udine, Italy 3Institute of Pathology, Faculty of Medicine, University of Ljubljana, Slovenia

4Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Brussel, Belgium

5Institute of Oncology, Ljubljana, Slovenia 6University Clinical Centre, Neurosurgery Clinic, Ljubljana, Slovenia

Cours International Medicine Genomique

October 17-21, 2016 Inst. Pasteur, Tunis

What is a brain tumor?

A brain tumor is an abnormal growth of tissue in the brain or central spine

that can disrupt normal brain function.

Benign tumors Malignant tumors

• The least agressive type.

• Do not contain cancer cells.

• Slowly growing.

• Clear borders.

• Do not spread into other

tissue.

• They contain cancer cells.

• They do not have clear borders.

• They grow rapidly.

• They invade surrounding brain

tissue.

Primary Metastatic

• They start in cells of the brain.

• They may spread to other parts

of the brain or to the spine.

• They spread rarely to other

organs.

• They begin in another part of the

body and spread to the brain.

• They are more common than

primary brain tumors.

SLOVENIA

150 of malignant brain tumors / year

60 glioblastoma / year

340 mio inhabitants

2 mio inhabitants

The most common primary brain tumors

All brain tumors Malignant brain

tumors

Meningiomas 34%

Gliomas 30% 70-80%

Pituitary adenomas 13%

Nerve sheath tumors 9%

Gliomas are the most common primary intracranial

tumor, representing more than 70% of malignant brain

tumors. Although relatively rare, they cause significant

mortality and morbidity. Glioblastoma multiforme (GBM)

is the most common and deadliest of malignant primary

brain tumors in adults and is one of a group of tumors

referred to as gliomas. The incidence, or the number of

new diagnoses made annually is 2 to 3 per 100,000

people in the United States and Europe. GBM accounts

for 12% to 15% of all intracranial tumors and 50% to

60% of all gliomas.

There are three types of normal glial cells that can produce tumors. An

astrocyte will produce astrocytomas (including glioblastomas), an

oligodendrocyte will produce oligodendrogliomas, and ependymomas come

from ependymal cells. Tumors that display a mixture of these different cells are

called mixed gliomas.

“Glioma” is a general term used to describe any tumor that arises from the

supportive (“gluey”) tissue of the brain. This tissue, called “glia,” helps to keep

the neurons in place and functioning well.

www.ABTA.org, 2013

The normal differentiation process originates three main types of cells in the mature

CNS: neurons, glial cells (oligodendrocytes, astrocytes, ...), and ependymal cells (not

shown). Malignant transformation to glioma occurs through didefferentiation process

either from glial cells or from glia progenitor cells and/or from neuronal stem cells.

Celine S. et al., INTECH, 2013

Symptoms

• Recurrent headaches

• Issues with vision

• Seizures

• Changes in personality

• Short-term memory loss

• Poor coordination

• Difficulty in speaking or compehending

Zhang P. et al, TIN, 2009

Cancer stem cells within a brain tumor can arise from normal stem cells or

from neural progenitor cells that harbor mutations. Cancer stem cells within

glioblastomas can be particularly resistant to cytotoxic therapies, and therefore

are a source of cells for cancer recurrence. To meet the challenge of a cure for

glioblastoma, new therapies that selectively suppress proliferation and/or kill

cancer stem cells must be found.

Characteristics and fates of normal neural stem cells and cancer stem cells.

http://www.google.si/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiT0K7K4oHOAhVPahoKHWExAtcQjRwIBw&url=http%3A%2F%2Fwww.eylea.com%2Feylea-insight%2Fkol-presentations%2Faflibercept-the-molecule%2F&psig=AFQjCNFv7T-tSL4ESsm809UAeXUNV0vvWg&ust=1469094626013857

Chromosomal region Type of alteration Candidate gliomas genes

1p36.31-pter Gains and deletions Not known

1p36.22-p36.31 Gains and deletions Not known

1p34.2-p36.1 Gains and deletions Not known

1q32 Gains Receptor interacting protein kinase (RIPK5)

Mouse double munite 4 (MDM4)

Phosphatidylinositol-4-phosphate-3-kinase

Catalytic subunit Type 2 beta (PK3C2B)

And others ...

4q Deletions NMA-related kinase (NEK1), NIMA

7p11.2-p12 Amplifications/gains Epidermal growth factor receptor (EGFR)

9p21-p24 Deletions Cyclin-dependent kinase inhibitor 2A (CDKN2)

10q23 Deletions Phosphatase and tensin homolog (PTEN)

10q25-q26 Deletions O-6-methylguanine-DNA-methyltransferase (MGMT)

11p Deletions Cyclin-dependent kinase inhibitor 1C (CDKN1C)

Related RAS viral oncogene homolog 2 (RRAS2)

12q13.3-q15 Amplifications Mouse double minute 2 homolog (MDM2)

Cyclin-dependent kinase 4 (CDK4)

And others ...

13p11-p13/13q14-q34 Loss Retinoblastoma 1 (RB1)

19q13 Loss Glioma tumor suppressor candidate region gene 1

GLTSCR2

DNA ligase I ATP dependent (LIG1)

Cytohexin 2 (CYTH2)

And many others ...

22q11.21-q12.2 Loss 28 genes incl. Integrase interactor 1 (INI1)

22q13.1-q13.3 Loss Not known

T.Mesti and J.Ocvirk: Radiol. & Oncol, 2016

The chromosomal alterations mostly observed in gliomas

Syndrome Gene name Chromosomal

location

Neurofibromatosis 1 Neurofibromin 1 (NF1) 17q11

Neurofibromatosis 2 Neurofibromin 2 (NF2) 22q12

Tuberous sclerosis Tuberous sclerosis 1 (TSC1)

Tuberous sclerosis 2 (TSC2)

9q34

16p13

Retinoblastoma Retinoblastoma 1 (RB1) 13q14

Li-Fraumeni

syndrome

Tumor suppressor p53 (TP53) 17p13

Turcot's syndrome

Multiple harmartoma

Adenomatous polyposis coli (APC)

DNA mismatch repair genes:

Recombinant human MutL homolog-1 (hMLH1)

MutS homolog 2 (hMSH2)

Mismatch repair endonuclease (PMS2)

Phosphate and tensin homolog (PTEN)

5q21

3p21.3

2p22-21

7p22

10q23.3

Inherited mutations present in patients with malignant gliomas

Differentiared astrocytes or precursor cells

p53 mutation (>65 percent) EGFR amplification (40 percent)

PDGF-A, PDGFR-alpha overexpression (60 %) overexpression (60 percent)

Low grade astrocytoma MDM2 amplification (<10 percent)

overexpression (50 percent)

LOH 19q (50 percent)

RB alteration (25 percent)

p16 deletion (30-40 percent)

Anaplastic astrocytoma

LOH 10p and 10q

LOH 10q PTEN mutation (30 percent)

PTEN mutation (5 percent)

DOC loss of expression (50 percent)

PDGFR-alpha amplification (<10 percent) RB alteration

Secondary glioblastoma Primary glioblastoma de novo

Development of

primary and

secondary

glioblastoma

T.Mesti and J.Ocvirk:

Radiol. & Oncol, 2016

Marker Protein type Function Prognostic

significance

CD 133 5-TM protein Only CD133+ cells may produce a

tumor. +++

- -

Nestin Filament marker in

neuronal progenitor

cells

Neuronal development /

dedifferentiation +++++

+/-

- -

CD 133 /nestin +

Podoplanin Mucin-type TMP Motility & invasion

spheroid formation ++

CD 15 Differentiation antigen Impl. of CD+ GB cells may produce

new tumors. + / -

A2B5 Surface glycoside A2B5 cells have CSC properties + / -

Musashi-1 Neuronal RNA-binding

protein

Neurosphere formation – CSC

properties ++ -

+/-

BM/1 Polycom-group protein Tumor suppressor pathway

regulator +/-

-

SOX2 Sox-family transcription

factor

Normal pluripotent cell development -

Id1 DNA-binding protein

inhibitor

Cell growth, senescence, and

differentiation / Proliferation, neo-

angiogenesis, invasion

++

Oct-4 Octamer-binding

transcription factor 4

Regulator of ESC self-renewal and

differentiation +

Dalhrot et al.: Int. J. Clin. Ex. Pathol., 2013

Review of twenty-seven independent studies on putative CSC markers in gliomas

• Overexpression

• Prognosis

• Prediction

• Survival

50 % 50 %

VHH:

• single domain, small size (15 kDa)

• high expression levels

• soluble and stable

• easy to handle compared to scFv

CAMELIDAE (camels, llamas) heavy chain ANTIBODIES

scFv VHH

V G L W

37 44 45 47

FY

E R VGLW

VL

VH

Covering of the hydrophobic patch by a long CDR3 allows soluble expression of VHH domains in the absence of VL counterpart.

Structural representation

Human VH camelid VHH

CDR1 CDR3

CDR2

Immunization: GBM whole cells enriched with GSC

Panning and screening

Cancer tissue / cells Normal tissue / cells

Glioblastoma tumor total protein extract Normal brain tissue protein extract

GSC membrane protein extract Normal brain tissue protein extract

GSC membrane protein extract NSC membrane protein extract

GSC total protein extract NSC total protein extract

GSC whole cells NSC whole cells

GSC whole cells NSC membrane protein extract

Ivana Jovčevska et al.: PLoS One, 24 November 2014 .

TRIM 28 Tripartite Motif

Transcription factor / KAP1,

KRIP1, TIF1β

SC: Maintaining

pluripotency.

SC: Differentiation.

AC: Anti-differentiation.

Overexpressed in cancer;

compex role, profiliferative...

ACTB Β actin Specifically controls cell

growth & migration & G

actin pool.

Mutant (unpolimerized)

actin in transcriptional

control.

AU (antigen) /

AU (GADPH)

Glioblastoma Glioblastoma Normal Glioblastoma Glioblastoma Normal

stem cells cells brain tissue stem cells cells brain tissue

Membrane fraction Cytosolic fraction Total extract

Nanobodies (Nbs) and the corresponding antigens obtained atfer several rounds of

phage-display VHH-library panning followed by MS analysis

Nanobody Antigen Name

Nb141 ACTB Beta actin

Nb237 TRIM28 Tripartite motif-containing 28 (TRIM28), also known as

transcriptional intermediary factor 1β (TIF1β) and KAP1

(KRAB-associated protein-1)

Nb10 NUCL Nucleolin

Nb79

VIM

Vimentin

Nb179

NAP1L1

Nucleosome assembly protein 1-like 1

Nb314

DPYSL2/CRMP1

MTHFD1

Dihydropyrimidinase-related protein 2/Collapsin response

mediator protein 1

Methylenetetrahydrofolate dehydrogenase 1

Nb394 DPYSL2/CRMP1 Dihydropyrimidinase-related protein 2/Collapsin response

mediator protein 1

Nb225

Nb206

TufM Tu translation elongation factor, mitochondrial

Nb395 ALYREF THO complex, subunit 4

In ed. p.: I. Jovchevska et al., 2016

Starting QVQL Ending TVSS

Unique CDR3s: binding different antigens

GLEW of Nb10: VH germline origin during V-D-J recombination

R50: VHH origin

Nanobody:Antigen binding

• Nb141:β-actin

GSC GBM

• Nb237:TRIM28 GSC GBM

* * Y Y * * * * Y Y * *

Y – staining with commercial antibodies after

incubation with Nb141 (anti-ACTB) Y – staining with commercial antibodies after

incubation with Nb237 (anti-TRIM28)

I. Jovčevska et al.: PLoS One, 2014

TufM:

Tu translation elongation factor,

mitochondrial

Mitochondrial translational elongation.

Reported to be involved in exosome formation and as a component

of natural killer cells.

A nuclear gene encoded protein, synthesized in

cytoplasm and imported to mitochondria.

Reported down-regulated in lung cancer cells.

Reported unfavorable prognostic indicator in CRC.

Reported elevated expression in GC.

HaCat Anti K14 stained (red) / DAPI stained (blue) U251 DAPI stained (blue)

Nb 206 – antiTufM

FITC-Nb 206 (green): sub-membrane localisation + close

to nuclei of dividing cells.

FITC-Nb 206 (green): centrosomal localisation in dividing cells.

Nb206, model from 4WGV

Arg103 Asp260

EF-TU, 1D2E

Nano body - Divalent ion transporter, 4WGV

In ed. p.: J. Stojan et al., 2016

Vimentin Major cytoskeletal component of mesenchymal cells.

Used as a marker of :

• mesenchymally-derived cells;

• cells undergoing an epithelial-to-mesenchymal

transition (EMT) during both normal development

and metastatic progression.

Formation of lamellipodia and invadipodia during cell migration and invasion.

Three studies already reported VIM over-expression in GBM.

Reported involved in different forms of cancers.

Reported involved in glioma metastatic abilities.

Protein expression data for ALYREF, CRMP1 and VIM.

Loading control, GAPDH, is included to show equal protein amount among samples.

Nucleolin An eukaryotic nucleolar phosphoprotein.

Involved in the synthesis and maturation of ribosomes.

Located mainly in dense fibrillar regions of the nucleolus.

Two NUCL types reported in gliomas: cytoplasmic and surface.

Centrosomal nucleolin is required for microtubule network organization.

Surface NUCL reported dependent on association with actin cytoskeleton.

Reported as regulator of glioma cell migration.

Reported that surface nucleolin increase with the malignancy grade of glioma.

DPYSL2 / CRMP2:

Dihydropyrimidinase-related protein 2/

Collapsin response mediator protein 1

Expressed during neuronal development, and rarely in adult brain.

Reported under-expressed in GBM.

Reported overexpressed in CRC and GC.

NAP1L1:

Nucleosome assembly protein 1-like 1 A member of the nucleosome assembly protein (NAP) family.

Locates within the nucleus of dividing cells.

Involved in cell cycle progression, transciptional regulation

and modification of chromatin structure.

Posible role in modulating chromatin formation.

Contribute to the regulation of cell proliferation.

In dividing cells influences expression of pro-proliferative genes.

Reported over-expressed in pancreatic neuroendocrine neoplasm

metastases.

Reported involved in lymphoblastic leukemia, ovarian cancer,

breast cancer, hepatoblastoma, CRC and lung cancer.

MTHFD1: Methylenetetrahydrofolate dehydrogenase 1

A protein with three distinct enzymatic activities in the interconversion

of 1-carbon derivatives of tetrahydrofolate, substrates for

methionine, thymidulate and de novo purine synthesis.

The folate metabolic system MTHFD1 is up-regulated in cancer

and is involved in rapid cancer cell proliferation.

Reported role of folate metabolism in the development of primary brain

tumors (meningioma and glioma):

genotypes associated with increased 5,10 methylenetetrahydrofolate

levels associated with elevated risk.

ALYREF:

THO complex, subunit 4

Nuclear protein which functions as a molecular chaperone.

Involved in transport of mature mRNA out of the nucleus.

Probably regulates dimerization, DNA binding, and transcriptional

activity of basic region-leucine zipper (bZIP) proteins.

The protein is a part of NUP107 complex which has been reported

altered in 19% of GBMs.

Correlations among the proteins studied with OncoFinder.

Blue color: proteins investigated in this study. Black arrows: interactions through intermediate compounds.

Grey color: other proteins involved in the interaction network. Blue arrows: direct activations a protein by the preceding protein.

Interaction map of top differential proteins identified by Nanobodies.

In ed. p.: I. Jovchevska et al., 2016

Histograms of area under curve (AUC) values

x-axis: AUC

y-axis: frequency

Red bars: genes of interest

Grey bars: overall distribution

A: Histogram of gene distribution analyzed

as cancer vs. normal.

It suggests DPYSL2 as a great,

and NUCL, TRIM28, VIM and NAP1L as

good markers for distinguishing cancer from

normal samples.

B: Histogram of gene distribution analyzed

as GBM vs. LGG.

NAP1L1, NUCL, CRMP1, ACTB and VIM

are pointed out as genes that can be used

as markers to distinguish between tumor

classes.

In ed. p.: I. Jovchevska et al., 2016

Genemania network

Number of surviving cells relative to control after 24 hours (Filip Mihalic, Mirjana Liovic, 2015).

0

0,5

1

1,5

2

untreated HaCat

α-β actin 10 μg/ml

α-β actin 100 μg/ml

α-206 10 μg/ml

α-206 100 μg/ml

α-Trim 28 10 μg/ml

α-Trim 28 100 μg/ml

HaCat

0

0,5

1

1,5

2

untreated TR146

α-β actin 10 μg/ml

α-β actin 100 μg/ml

α-206 10 μg/ml

α-206 100 μg/ml

α-Trim 28 10 μg/ml

α-Trim 28 100 μg/ml

TR146

0

0,5

1

1,5

2

untreated U251

α-β actin 10 μg/ml

α-β actin 100 μg/ml

α-206 10 μg/ml

α-206 100 μg/ml

α-Trim 28 10 μg/ml

α-Trim 28 100 μg/ml

U251

a-TRIM28/a-beta_actin nanoparticles have a greater effect on

glioblastoma cells

Wst-1 proliferation test on glioblastoma cell lines

Nb 206: anti-TufM

Nb 237: anti-TRIM28

Neja Zupanec et al., 2015

Wst-1 proliferation test on glioblastoma stem

cells (NCH 644 / NCH 421K) vs. neural stem

cells (NSC)

Archeosomes

https://youtu.be/P18fSQuCjNM?t=14

https://www.youtube.com/watch?v=P18fSQuCjNM

GPMV – Giant Plasma Membrane Vesicle formation

HACAT

TR146 fluorescenčno: GPMW-1

GPMW-2

In ed. p.: M.Liovic et al., 2016

ACKNOWLEDGMENTS:

Applied Biological

Sciences & Engineering,

Vrije Universiteit Brussel,

Brussel, Belgium

Serge Muyldermans

Medical Centre for

Molecular Biology,

MF UL

Ivana Jovčevska

Neja Zupanec

Mirjana Liović

Nina Kočevar

Damjana Kastelic

Helena Klavžar

Radovan KOMEL

et al.

AOUD

Udine, Italy

Daniela Cesselli

Institute for Pathology,

Faculty of Medicine UL

Clara Limbaeck Stokin

DKFZ

Heidelberg, Germany

Damjana Kastelic

•Slovenian Research Agency

• Interreg GLIOMA

• Ad-Futura Science foundation

• VUB, Brussel

• Babraham, Cambridge, U.K.

• CNRS, Gif-sur-Yvette, France

NIB

Ljubljana

Tamara Lah Turnšek

Neža Podergajs

Martina Mršnik

Elettra Synchrotron

Trieste, Italy

Paola Storici