Investigation of the Role of Chondroitin Sulfate Proteoglycan in Causing Treatment Resistance in Melanoma to PI3k/mTOR Inhibition

A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL

OF THE UNIVERSITY OF MINNESOTA BY

Sehrish Javaid

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

Arkadiusz Dudek

July 2012

© Sehrish Javaid 2012

i

Acknowledgements I sincerely thank Dr.Arkadiusz Dudek for giving me an opportunity to work in his lab. His guidance and trust has made this final defense possible. I would also like to thank Dr.Jim McCarthy for his support and valuable suggestions for my research and Dr.Raj Gopalakrishnan for reviewing my work and helping me throughout the program. I am also very thankful to Kaoru Terai for teaching me the basics in the lab. I respectfully extend my appreciation to the members of McCarthy lab, especially Matt Price and Leah C. Wanshura for their patience, support and contribution to my work.

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Dedication This thesis is dedicated to the loving memory of my mother, who taught me to be strong, persevere and never give up. My father, siblings and rest of the family who supported my decision to pursue research. My husband, for putting up with my doing this and encouraging me to work hard, I could not have done this without his love and support.

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Abstract

Melanoma is one of the deadliest tumors. Despite the advances in understanding the

melanoma cell biology and drug discoveries, treatment resistance remains a challenge.

Chondroitin sulfate proteoglycan (CSPG4) is expressed on the surface of melanoma

cells, and its role in metastatic progression has been established. One known mechanism

responsible for this activity is modulation of the activity of ERK1,2. PI3k/mTOR

pathway is also frequently deregulated in melanomas . We therefore investigated if

CSPG4 has an impact on PI3K/mTOR pathway activity. Our results indicate that

CSPG4 causes resistance to growth inhibition and apoptosis in early stage melanoma

cell lines. The knockdown of CSPG4 sensitized the advanced disease stage cell lines to

growth inhibition. CSPG4 also induced resistance at various levels of mTOR pathway

in different cell lines, and resulted in inhibition of motility and invasion in response to

treatment with PF-05212384, a potent PI3K/mTOR inhibitor. Melanoma cell lines

derived from all stages of melanoma growth showed resistance to inhibition of 4ebp1.

Our results indicate that CSPG4 play a role in decreased sensitivity of melanoma cells

to mTOR pathway inhibition.

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Table of Contents

Acknowledgements………………………………………………………………………i

Dedication……………………………………………………………………………….ii

Abstract…………………………………………………………………………………iii

List of Tables…………………………………………………………………………….v

List of Figures…………………………………………………………………………..vi

Introduction……………………………………………………………………………...1

Materials and Methods…………………………………………………………………..7

Results………………………………………………………………………………….13

Discussion………………………………………………………………………………31

Conclusion……………………………………………………………………………...35

Bibliography………………………………………………………………...………….36

v

List of Tables

TABLE 1: Characteristics of melanoma cells used for study…………………………7

vi

List of Figures

FIGURE 1. The PI3k/mTOR pathway………………………………………………….3

FIGURE 2. Structure of the TRIPZ shRNAmir……………………...…………………8

FIGURE 3. Validation of shRNA knockdown of CSPG4 in WM1341D and 1205LU...9

FIGURE 4. CSPG4 provides protection to anti proliferative effects of PF- 5212384…..

………………………………………………………………………………………….14

FIGURE 5. CSPG4 causes cells to be less responsive to the inhibition of

motility…………………………………………………………………………………20

FIGURE 6. CSPG4 causes the cells to be more invasive and resist the inhibition of

invasion…………………………………………………………………………………23

FIGURE 7. The presence of CSPG4 reduces apoptosis in early growth phase

melanoma cell lines……………………………………………………………….……24

FIGURE 8. The expression of CSPG4 causes preservation of mTOR downstream

signaling………………………………………………………………………..………27

FIGURE 9. Proposed model of resistance to PI3k/mTOR inhibition in the presence of

CSPG4……………………………………………………………….…………………33

1

Introduction:

Melanoma is the 6th most common cancer diagnosed in U.S. It is the second most

common cancer diagnosis in women from birth to age 39 years, and third most common

diagnosed in men in the same age group (Siegel et al 2012). Melanoma is caused by the

malignant changes in the melanocytes, which are responsible for producing melanin in

skin. The incidence of cutaneous malignant melanoma has increased over the last

several years by 3-7 % for fair skin Caucasian population (Lens, M. B. and M. Dawes

2004). The mortality from the disease has also increased throughout the world in the

past few decades.

Melanomas have a multifactorial etiology. Different environmental, genetic and host

factors contribute to the establishment of the disease. The major environmental

contributing factor is the exposure to the sunlight. People living close to equator or

coastal areas have an increased chance of getting the disease (Oliveria et al 2006). The

presence of acquired dysplastic nevi increases the risk of getting the disease (Bauer et

al). Patients with melanoma may also have a family history of the disease, as some of

the melanoma associated mutations maybe inherited which causes a significant increase

in the risk of melanoma. (Avils, Jos and Pablo Lzaro 2006)

Malignant melanoma is considered to be the most therapeutically challenging

malignancy. The prognosis for patients with high risk or advanced metastatic disease

remains poor (Eggermont, A. M 2010). Patients with advanced disease and multiple

metastases have a median survival of less than one year. The standard treatment for

early stage disease is surgical resection followed by adjuvant therapy. Dacarbazine is

used most commonly to treat the advanced cases of melanoma (Garbe, C. et al 2011).

The response rate has been reported to be 5-12%. The rate of relapse with standard

chemotherapy is high as the results achieved with this treatment are transient. Local

radiation therapy has also been used. Immunotherapy, especially with high dose IL-2

has resulted in durable response in 10% of patients(Zito et al 2012). A combination of

2

different chemotherapeutic agents failed to show promising results in clinical trials

(Davies, Michael A, et al 2012).PLX4032 (Vemurafenib) a BRAF inhibitor showed an

improvement in the rate of overall survival, however resistance emerged after initial

response(Flaherty, Keith T, et al 2010).

Melanoma is characterized by aberrations in multiple cellular pathways. The

RAS/RAF/MEK/ERK pathway is one of the most well studied pathways involved in

melanoma progression (Dhomen, Nathalie, and RichardMarais 2009). RAS mutations

activate downstream RAF (CRAF,BRAF)which ultimately activates MEK/ERK that

controls the cell proliferation and survival. ERK is hyper activated in 90% of human

melanoma (Arkenau, H-T et al 2011).

The PI3k/AKT/mTOR pathway regulates important cellular processes like growth,

differentiation, survival, apoptosis and migration. The PI3ks are family of lipid kinases,

that phosphorylate phosphatidylinositol diphosphate (PIP2) to its corresponding

phosphatidylinositol triphosphate (PIP3). PIP3 acts as a second messenger and activates

AKT, a serine/threonine kinase, which is the central mediator of the PI3k

pathway(Figure 1). AKT activates mTOR through PRAS40, which promotes protein

synthesis and cell growth. The mTOR is composed of two complexes. Activation of

rapamycin sensitive mTORC1 leads to increased p70 S6 kinase activity and increased

protein synthesis by phosphorylation of eukaryotic initiation factor 4E.The mTORC2

complex completes the AKT activation by phosphorylating AKT. AKT also influences

cellular proliferation by inactivating cell cycle inhibitors (p27 and p21) and promoting

cell cycle proteins. AKT also inhibits pro-apoptotic genes and inhibits apoptosis. PTEN

regulates this pathway by antagonizing the activity of PI3k (Willems, Lise, et al 2012).

The components of the PI3k/AKT/mTOR pathway became target for cancer

therapeutics. The inhibitors of these pathways inhibit cell proliferation; decrease

survival and also effect angiogenesis and metastasis (Brachmann, Saskia, et al 2009). A

number of these compounds have entered phase 1 clinical trials. The compounds are

3

still in the process of evaluations, however the early data has shown promising results.

(Courtney et al 2010).

FIGURE 1. The PI3k/mTOR pathway

PI3k regulates PIP2/PIP3, which phosphorylates AKT. PRAS40 downstream of

AKT phosphorylates mTOR which further effects S6k and 4ebp1.

One strategy to improve the treatment outcome using these inhibitors was to target the

PI3k and mTOR complexes simultaneously, to prevent the reactivation of AKT as seen

after treatment with rapamycin and its analogues (Mazzoletti, Marco et al 2011).

The PI3k/AKT/mTOR pathway is also hyper activated in majority of melanomas

(Davies, M. A. 2012). The PI3k/AKT/mTOR pathway can be unregulated due to several

upstream mutations, e.g. PI3k or AKT amplification and PTEN deletion. PTEN, which

regulates the activation of PI3k and AKT, is deleted in 12% of melanomas (Yajima,

Ichiro et al 2012). There can be activation or amplification of genes encoding PI3k or

4

AKT.AKT amplification has been identified in 40-60% of melanomas (Amaravadi, R.

K. and K. T. Flaherty. 2007).

PF-5212384 (PKI-587) is a dual PI3k/mTOR inhibitor. It is a selective, ATP-

competitive, and reversible PI3K/mTOR inhibitor (Venkatesan et al). The inhibitor

displayed antitumor activity in breast, colon, gliomas, and non–small cell lung cancer

xenograft models. It has been advanced to phase 1 clinical trials. The IC50 values for

the various isoforms of PI3K and its frequent mutants varied from 0.0004 to 0.008 µM.

The IC50 for the mTOR complex was 0.001 µM (Mallon, Robert et al 2011).

Chondroitin sulfate proteoglycan-4 (CSPG4), (also known as high molecular weight-

melanoma associated antigen, melanoma chondroitin sulfate proteoglycan) is a

membrane bound protein, covalently modified by the chondroitin sulfate

glycosaminoglycan, having a diverse expression in the human tissues (Campoli,

Michael et al 2010). It was first identified 30 years ago. It is expressed in normal

melanocytes, pericytes, and endothelial cells. It is also present in the multipotent stem

cells in the human bone marrow. It may have an important role in the development and

maintenance of the healthy tissues. It is expressed in oligodendrocytomas, gliomas,

triple-negative breast carcinomas, and squamous cell carcinoma (Wang, Xinhui, et al

2010). It is highly expressed by human melanoma cells. This tumor antigen plays an

important role in melanoma cell biology by modulating proliferation, cell adhesion, and

motility (Wang, Jian et al 2011).

Melanoma cells transfected with CSPG4 cDNA displayed higher migratory ability than

parental cells. CSPG4 expression in radial growth phase (RGP) human melanomas

facilitates migration, protease activation, and epithelial to mesenchymal transition.

CSPG4 facilitate sustained, high-level activation of key survival and growth pathways,

in particular focal adhesion kinase (FAK) and ERK 1,2 pathways (Yang, J et al 2004).

Recently it was reported that the inhibition of CSPG4 using monoclonal antibody

enhanced and prolonged the effect of PLX4032, a selective BRAF inhibitor.

5

Combination of CSPG4 monoclonal antibody with PLX4032 inhibited the cell growth

than either agent alone and delayed the development of resistance in treated cells. The

combination of the treatment inhibited multiple signaling pathways (Yu, L et al 2011).

The exact mechanism by which the CSPG4 modulates the tumor biology is not known.

CSPG4 does not have any known endogenous catalytic activity. Evidence indicates that

they participate in signal transduction by acting as co-receptors working with other

receptors containing intrinsic receptor tyrosine kinase activity. They can also interact

directly or indirectly with FAK and ERK1/2. CSPG4 also interacts with ECM

components linking the tumor microenvironment with various signaling

pathways(Price, M. A et al 2011). Most of the efforts to investigate the role and the

potential mechanism of action have focused on the FAK and the MAPK pathway.

Due to the high prevalence of BRAF and NRAS mutations, and the clinical outcome of

the BRAF inhibitor therapy, there has been a strong interest in the RAS-RAF-MEK-

ERK signaling pathways in melanoma. The PI3k-AKT-mTOR pathway is also activated

in melanomas and may play an important role in the pathogenesis of the disease

(Haluska, Frank, et al 2007). Sinnberg et al (2009) reported that the pharmacological

inhibition of AKT pathway sensitized the melanoma cells to cisplatin and

temozolomide. Co-treatment of melanoma cells with AKT pathway inhibitors and

chemotherapeutic led to a 2 to 3 fold increase of apoptosis. The individual inhibition of

mTOR and PI3k by rapamycin and wortmannin slightly repressed cell growth in

monolayer culture; however their combination with cisplatin or temozolomide

significantly increased growth inhibition in metastatic melanoma cell lines. The

combination also abolished invasive tumor growth of melanoma cells in organotypic

culture. Studies using BRAF mutant and PTEN null melanoma cell lines have indicated

that these cell lines show cell cycle arrest in response to the BRAF or MEK inhibition,

while the cell lines harboring BRAF mutations and a normal PTEN undergo apoptosis

(Paraiso, Kim H T, et al 2011).

6

Our aim for this study was to investigate the effect of CSPG4 on inhibition of PI3k-

AKT-mTOR pathway using PF-5212384 (PKI-587) in melanoma cell lines.

7

Material and methods:

Cell lines:

The following cell lines from Wister Institute were used for our study:

TABLE 1: Characteristics of melanoma cells used for study

Site CSPG4

Status

PTEN BRAF

WM1552C RGP - Mutated/Deleted V600 Activating

mutation

WM1341D VGP + Wild type V600 Activating

Mutation

1205LU Metastatic + Mutated/Deleted V600 Activating

mutation

Information adapted from the Wister institute’s website.

WM1552C CSPG4 expressing stable transfectant:

CSPG4 expressing WM1552C were generated previously by Yang et al and provided

by Dr. McCarthy’s lab for our project. Briefly, Full length human CSPG4 cDNA was

generated by RT-PCR amplification of mRNA isolated from A375SM cells. WM1552C

cells were transfected with vector containing full length CSPG4 and puromycin

resistant gene. Transfected cells expressing CSPG4 were selected for stable

transmission of vector containing CSPG4 with puromycin.

8

CSPG4 Knockdown:

CSPG4 positive 1205LU and WM1341D cell lines were transduced by Dr. McCarthy’s

lab with inducible shRNA against CSPG4 (TRIPZ shRNAmir). The transduced cell

lines were selected and maintained in 0.5ug/ml puromycin in growth media.

Doxycycline at a concentration 1ug/ml was introduced in the media for 48 hours to

induce the expression of the shRNA.

FIGURE 2. Structure of the TRIPZ shRNAmir

The pTRIPZ vector is engineered to provide the expression of shRNA in the

presence of doxycycline, which was added at a concentration of 1ug/ml in the

media. The TRIPZ vector has two regulatory components:

Tetracycline response element (TRE), and transactivator.

The TRE is composed of operator fused to CMV minimal promoter and binds to

transactivator. The transactivator binds and activates the expression from TRE

promoters in the presence of doxycycline. It only binds to the TRE in the presence

of doxycycline and certain mutations increases its sensitivity to doxycycline.

Information and image adapted from Thermo Scientific website

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FIGURE 3. Validation of shRNA knockdown of CSPG4 in WM1341D and 1205LU

The expression of CSPG4 was knocked down using shRNA against CSPG4 in

WM1341D and 1205LU. The protein expression was analyzed using western blot.

Significant reduction in the protein expression was observed after the introduction

of anti CSPG4 shRNA compared to control. The reduction of expression was more

pronounced in WM1341D compared to 1205LU.

Image provided by Leah C. Wanshura

Cell proliferation assay:

Cell proliferation assay were performed as described previously (Paraiso et al 2010).

Briefly, cells were seeded in triplicates at a density of 3 x 104/ml in 96 well plates.

Media was replaced next day with media containing inhibitor and cells were incubated

for 72 hours. MTT assay from Roche was performed according to the manufacturer’s

instruction. 3(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide(MTT) was

added to the media at a final concentration of 0.5mg/ml and incubated for 4 hours at 37

C .Equal volume of solubilization solution was added and the plates were incubated

overnight to solubilize the crystals. Absorbance was read at 562nm.

Wound healing assay:

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Tissue culture plates were coated with fibronectin at a concentration of 1ug/ml

overnight. Extra fibronectin was removed the next day and the plates were allowed to

air dry. Ibidi cell culture inserts were attached. Cells were seeded at a density of 5 X

105/ml in the insert according to the manufacturer’s protocol. Cells were allowed to

become confluent overnight and treated with the inhibitor next day. Pictures were taken

at indicated time points. Gimp freeware was used to quantify the area covered by the

cells by calculating the pixels.

Cell Invasion assay:

Cells were serum starved for 24 hours. BD Biocoat matrigel invasion chamber inserts

were rehydrated with serum free media for 2 hours at 37 C. Cells were lifted using

5mM EDTA for five minutes and washed thrice before resuspending at a cell density of

1 x106 /ml. Cells were then either left untreated or treated with the inhibitor for 30

minutes at 37 C with shaking. 500uL of the cell suspension was placed in the matrigel-

coated insert. 750uL of media containing 10% serum was added to the bottom well to

add as chemoattractant. The chambers were incubated for 4 hours at 37 C. The inserts

were removed from the chamber and excessive media and cells were removed with a

cotton swab. The inserts were then fixed with methanol for fifteen minutes at room

temperature and stained with crystal violet (Shaw, Leslie M.). Images were taken at

40X magnification.

Western blot analysis:

11

Cells were plated at a density of 150,000 cells/ml in six well plates. After 48 hours, they

were treated with inhibitor at indicated doses for 6 hours. Cell lysate were collected as

described previously (Sinnberg et al 2009). Cells were washed twice with ice cold PBS.

Phosphosafe extraction reagent was used to lyse the cells for five minutes at room

temperature according to manufacturer’s instruction. The lysates were cleared by

centrifugation for 5 minutes at 14,400 x g at 4 C. BCA protein assay was performed to

quantify the amount of protein and 30ug protein was used to load the gels. The samples

were subjected to SDS-PAGE and transferred to PVDF membrane. The membrane was

blocked with 5% milk in TBST and washed with TBST for fifteen minutes thrice before

probing with the antibodies overnight. HRP conjugated secondary antibody was then

used to detect the primary antigen. The membrane was immersed in Pierce Supersignal

West Dura chemiluminescent substrate for five minutes and then exposed to X-ray

films. Image from NIH was used to perform the densitometry. The following antibodies

from Cell Signaling Technology (Beverly, MA) were used: Phospho-P70

s6k(9234)(Thr389)1:1000, Phospho-PRAS (2997)1:2000,Phospho-4ebp1(2855) 1:1000

, P70 s6k(9202) 1:1000, 4ebp1(9452) 1:1000, Cleaved PARP(5625) 1:1000, Total

PARP (9542) 1:1000. Alpha Tubulin antibody(SC-8035) 1:1000, anti-rabbit HRP-IgG

secondary antibodies. (Jackson ImmunoResearch, West Grove, PA)1:10000.

PF-5212384:

PF-5212384 was obtained from Pfizer (New York, NY) under Material Transfer

Agreement. Also known as PKI-587, this is a highly potent dual PI3k/mTOR inhibitor.

12

The molecular weight is 615.73. Stock solution were made in DMSO at 4mM

concentration and used for further studies.

13

Results:

PF-5212384 inhibits the proliferation of melanoma cells:

To see the effect of the inhibition of the PI3k/mTOR by PF-5212384 on cell

proliferation, MTT assays were performed. We selected CSPG4 deficient WM1552C

and CSPG4 expressing 1205LU and WM1341D parental cell lines. The cells were

treated with the inhibitor for 72 hours as described previously.

The percent cell viability at the end of the assay for the CSPG4 deficient WM1552C

was 19.07 compared to 35.76 and 47.94 for CSPG4 expressing WM1341D and 1205LU

respectively when treated with 10uM dose (Figure 4A). The experiments were

performed in triplicates and repeated thrice. The difference between percent viability for

1205LU and WM1552C and WM1341D and WM1552C was statically significant (P<

0.05) at concentrations equal or higher than 1000 nM. The results indicate that the

CSPG4 expressing parental cell lines are less sensitive to the treatment with inhibitor

compared to the CSPG4 deficient cell lines.

The expression of CSPG4 protects cells from the effect of PI3k/mTOR inhibition:

When we treated the CSPG4 expressing WM1552C cell line with the PF-5212384

inhibitor for 72 hours, the percent cell viability at 10uM dose was increased to 65.96%

compared to 13.43% for controls (Figure 4B). The experiment was performed in

triplicates and the mean was statistically significant (P<0.05) at 1000 and 10000 nM.

The results indicate that the expression of CSPG4 on WM1552C causes them to resist

growth inhibition in response to treatment with the inhibitor.

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FIGURE 4. CSPG4 provides protection to anti proliferative effects of PF- 5212384.

Cells were treated with inhibitor for 72 hours and MTT assays performed. Data

represents cell viability as percent of DMSO control and the mean absorbance

value.

(A) CSPG4 expressing melanoma cell lines (1205LU and WM1341D) are less

sensitive to growth inhibition compared to CSPG4 deficient WM1552C. Data

represents mean of three independent experiments ± SD *, The difference between

the cell viability at this dose between the CSPG4 expressing and deficient cell line

was statistically significant (P<0.05) ; **, The difference between the CSPG4

expressing and deficient cell lines was significant(P<0.05).

15

(B) The expression of CSPG4 causes the WM1552C to be less sensitive to growth

inhibition. The percent cell viability was increased to 65.69% compared to 13.43

for controls. *, The difference between the cell viability at this dose between the

CSPG4 expressing and deficient cell line was statistically significant (P<0.05) (C)

Knockdown of CSPG4 in WM1341D sensitized them to the growth inhibition in

16

response to treatment (D) Knockdown of CSPG4 sensitized 1205LU cells to the

lower dose of inhibitor. *, The difference between the cell viability at this dose

between the CSPG4 expressing and deficient cell line was statistically significant

(P<0.05)

(E, F) Cells were serum starved and then treated with the inhibitor in the absence

or presence of 2% serum. No significant difference of growth inhibition between

CSPG4 expressing and deficient cell lines was observed (G,H) Serum starved cells

17

WM1552C cell with and without CSPG4 were stimulated with IGF-1 for 4 hours

and treated with inhibitor in the absence of serum. No difference in growth

inhibition was observed.

Knockdown of CSPG4 increased growth inhibition in CSPG4 positive 1205LU and

WM1341D:

To see if the knockdown of the CSPG4 increases the sensitivity of the cells to the

inhibition of proliferation, we induced the expression of shRNA against CSPG4 in

1205LU and WM1341D and treated them with the indicated doses. We observed no

difference in the growth inhibition at higher doses in the metastatic 1205LU cell line

with and without CSPG4 (Figure 4D). However at 100 nM the CSPG4 deficient cells

showed a growth inhibition of 19.79% compared to none for the CSPG4 expressing

controls. The difference between the mean was statistically significant. This result

indicates that in the metastatic cell line, the presence of CSPG4 does not provide

protection at higher doses, however when treated at lower doses, the CSPG4 expressing

cells were less sensitive to the inhibitor.

The percent cell viability for the WM1341D after the knockdown of CSPG4 was 26.30

compared to 50% for controls at 10uM drug treatment indicating an increased

sensitivity of the CSPG4 deficient cell lines to treatment (Figure 4C).

CSPG4 does not provide protection from the effect of treatment with PF-5212384

in the absence or low serum conditions:

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We wanted to evaluate whether CSPG4 would provide any protection from the effects

of the inhibition of PI3k/mTOR pathway in response to treatment with inhibitor in the

absence or decreased availability of serum. WM1552C cells were plated in 96 well

plates as described previously and serum starved for 24 hours. Inhibitor at the indicated

dose was added to media containing 2% or no serum. MTT assay was performed after

48 hours. No significant difference between the % cell viability for cells with and

without CSPG4 was seen (Figure 4E, F) indicating that CSPG4 does not provide

protective effects to the inhibition of PI3k/mTOR in the low serum condition.

CSPG4 does not provide protection from the inhibition of PI3k/mTOR inhibition

in response to IGF-1 stimulation:

We also wanted to see if CSPG4 would provide any protection from the inhibition of

cell proliferation when IGF-1 receptor upstream of PI3k (Pollak, Michael 2012) is

stimulated under low or no serum conditions. Cells were serum starved or treated with

2% serum for 24 hours after plating. IGF-1 was added at a concentration of 10 and

100ng for four hours, after which the media was replaced with fresh media containing

inhibitor. MTT assay was performed after 48 hours. We didn’t observe any significant

difference in the % cell viability for the CSPG4 expressing and deficient WM1552C

cell line (Figure 4G,H) indicating that the CSPG4 does not provide protection from the

inhibition of stimulated IGF-1 in the absence of serum.

CSPG4 Increase the cells resistance to the inhibition of motility in response to the

PI3k/mTOR inhibition:

19

Recent work has shown that the PI3k/mTOR pathway plays a critical role in regulating

the cancer cell motility and metastasis. Rapamycin, an inhibitor of mTORC1 complex

in addition to inhibiting protein synthesis and cell proliferation also affects the motility

of cells. Rapamycin regulates the motility of cells through the S6K and 4ebp1 pathway

(Zhou, Hongyu et al 2011). To see whether CSPG4 would provide any protection

against the inhibition of motility mediated by mTOR in response to treatment with PF-

5212384 we performed wound-healing assays. Tissue culture plates were coated with

fibronectin and a 500uM gap was created using Ibidi (Verona, WI) cell culture inserts.

Cells were grown to complete confluency and allowed to migrate over the gap. We

selected 75 nM dose of PF-5212384 in this experiment, because higher doses induce

cell apoptosis.

We first performed this experiment with the WM1552C cell line. Overall the CSPG4

expressing cells moved faster across the gap over a period of 24, 48 and 72 hours. They

covered 76% area of the total gap compared to 64% for the control after 72 hours

(Figure 5A,E). After the addition of inhibitor the CSPG4 expressing cells covered 43%

area of the initial gap. The reduction in the cell motility was more pronounced in the

CSPG4 deficient cells with only 31% area of the gap being closed at the end of 72 hours

(Figure 5B,E).

We also performed this assay for the metastatic 1205LU cell line after knocking down

the expression of CSPG4. The CSPG4 deficient cells were slow to move across the gap.

The most significant difference was seen at the end of 48 hours where the CSPG4

20

expressing cells covered 61.93% of the gap compared to 27% for the CSPG4 deficient

1205LU(Figure 5C,F). The rate of migration was reduced with the addition of inhibitor,

with the most difference seen at the end of 48 hours (Figure 5D,F), where the CSPG4

positive cells covered 52.64% more area of the gap compared to the CSPG4 deficient

cell line.

FIGURE 5. CSPG4 causes cells to be less responsive to the inhibition of motility

when treated with PF-5212384. A 500uM gap was created over fibronectin coated

tissue culture plates. Cells were allowed to migrate over a period of 72 hours with

or without serum.

(A) WM1552C expressing CSPG4 moved faster and covered 76% area of the

initial gap compared to 64% for controls after 72 hours. (B) The addition of

inhibitor reduced the migration in CSPG4 deficient cell lines to 31% compared to

43% for control after 72 hours.

21

(C) CSPG4 expressing 1205LU cells covered 61.93% gap area compared to 27%

for CSPG4 deficient cell lines at the end of 48 hours (D) After the addition of

inhibitor the CSPG4 expressing cells covered 52.64% more area of the gap

compared to the CSPG4 deficient cells.

(E) Photomicrograph of the migration of WM1552C with and without CSPG4

taken over a period of 72 hour

1

E  

2

22

(F) Photomicrograph of the migration of 1205LU with and without CSPG4 taken

over a period of 72 hour

This result indicates that the presence of CSPG4 provides resistance to complete

inhibition of migration/motility in response to PI3k/mTOR inhibition.

CSPG4 causes the cells to be more invasive and resist the inhibition in response to

the treatment with inhibitor:

Since it is impossible to tell the difference between migration and proliferation over a

period of more than 48 hours, we performed invasion assay using Matrigel coated

invasion chambers. Cells were serum starved for 24 hours. Cells were lifted off the plate

with EDTA to ensure that the surface domains of CSPG4 are not damaged. Cells were

treated with the inhibitor for 30 minutes in serum free media. Media containing 10%

serum was added as chemo attractant to the lower part of the invasion chamber. Cells

F  

23

were incubated in the chamber for four hours. The cells that had migrated to the bottom

of the insert were counted after staining. The CSPG4 expressing WM1552C were more

invasive compared to the CSPG4 deficient cells (Figure 6B). The difference between

the control and inhibitor treated cells was statistically significant. Similarly the CSPG4

deficient 1205LU were less invasive compared to controls (Figure 6A). The CSPG4

expressing cells were also less sensitive to the inhibition with more cells invading the

bottom of chamber compared to the CSPG4 deficient cells.

FIGURE 6. CSPG4 causes the cells to be more invasive and resist the inhibition of

invasion when treated with PF-5212384. Cells were treated with inhibitor and

incubated in BD matrigel coated invasion chambers. After four hours cells that

migrated to the lower side of the chamber were counted.

(A) The knockdown of CSPG4 in 1205LU made them less invasive. There were

also fewer invasions in presence of PI3K/mTOR inhibitor in the absence of

CSPG4. (B) The expression of CSPG4 in WM1552C caused them to be more

24

invasive. The reduction in invasion was less evident in the presence of CSPG4.

Data represent mean (n=3)±SD.*; The difference was statistically significant

between CSPG4 deficient control and inhibitor treated cells.

These experiments confirm that the expression of CSPG4 causes the cells to become

more invasive and motile, and resist the inhibition when treated with the PI3K/mTOR

inhibitor.

CSPG4 provides protection against apoptosis:

WM1552C cell line with and without CSPG4 was treated with the inhibitor for six hour.

Cleavage of PARP, which indicates apoptosis, was increased two-fold in the cell lines

deficient for the expression of CSPG4 (Figure 7a and 7b) .This indicates that CSPG4

provides resistance to apoptosis.

FIGURE 7. The presence of CSPG4 reduces apoptosis in early growth phase

melanoma cell lines. WM1552C with and without CSPG4 was treated with

inhibitor for 6 hours before collecting cell lysate. Western blot was performed to

detect the cleavage of PARP

A

25

(A) Western blot analysis of cleavage of PARP (B) Quantitative densitometry when

normalized to total PARP the C-PARP expression was increased two-fold at doses

higher than 300nM

CSPG4 preserves mTOR downstream signaling.

Next we wanted to evaluate if the presence of CSPG4 causes a resistance to the

inhibition of various signaling molecules in the PI3k/mTOR pathway. Cells were

treated with the inhibitor for 6 hours before preparing the cell lysate and performing

western blots. We selected PRAS downstream of AKT and p70s6k and 4ebp1

downstream of mTOR as our targets of interest.

The CSPG4 expressing radial growth phase cell line WM1552C was less sensitive to

the inhibition of 4ebp1 compared to the CSPG4 deficient WM1552C (Figure 8A, 8B).

The metastatic 1205LU cell line resisted complete deactivation of 4ebp1 even at 3000

nM (Figure 8A, 8D). However the CSPG4 expressing 1205LU had more 4ebp1

phosphorylation compared to CSPG4 deficient cells. The vertical growth phase cell line

WM1341D also had preservation of phosphorylation of 4ebp1 at 1000 nM

26

concentration of PF-5212384 in the presence of CSPG4 in contrast to the CSPG4

deficient cell line (Figure 8A, 8C).

The metastatic 1205LU cell line showed complete deactivation of P70sk at higher dose

of PF-05212384 in both the presence and absence of CSPG4 versus other melanoma

cell lines. The CSPG4 expressing cells however showed more activity of P70sk at

300nM compared to the CSPG4 deficient cells (Figure 8E, 8G). The activity of P70sk

was increased in the presence of CSPG4 in the WM1552C. The activity was completely

lost at 300 nM in the CSPG4 deficient cells however the CSPG4 expressing cells

showed resistance to complete deactivation (Figure 8E, 8F).

PRAS40 is a downstream target of AKT that binds to protein 14-2-3 and mTOR (Wang

et al 2012). In the metastatic 1205LU cell line, it resisted complete deactivation even at

3000 nM of PF-5212384. However the activity was decreased in response to the

treatment with inhibitor in the absence of CSPG4 (Figure 8H,8J). The same result was

achieved in the WM1341D where the CSPG4 expressing cells resisted complete

deactivation of PRAS compared to the CSPG4 deficient cells (Figure 8H,8I). These

results indicate that CSPG4 causes the PI3k/mTOR pathway to be less sensitive to

inhibition in response to treatment with the inhibitor.

27

FIGURE 8. The expression of CSPG4 causes preservation of mTOR downstream

signaling. Cells were treated with inhibitor for 6 hours before western blotting.

A

(A) Western blot analysis for the phosphorylated and total 4ebp1. After treating

with the inhibitor for 6 hours the phosphorylation of 4ebp1 was decreased more in

the absence of CSPG4. The vertical growth phase WM1552C cells were most

sensitive to the inhibition, while the metastatic 1205LU resisted complete

deactivation even at maximum dose.

28

Quantitative densitometry when normalized to total protein (B) WM1552C

showed increased 4ebp1 phosphorylation at 300nm in the presence of CSPG4 (C)

WM1341D showed 4ebp1 phosphorylation at 1000nM in the presence of CSPG4

(D) 1205LU showed an overall increase in activity of 4ebp1 in the presence of

CSPG4.

EA

29

(E) Western blot analysis for P70sk6k. The phosphorylation of the P70s6k is

affected differently in different melanoma cell lines.

Quantitative densitometry when normalized to total protein (F) WM1552C were

less resistance to phosphorylation at 300 nM in the presence of CSPG4 (G) 1205LU

showed resistance to dephosphorylation in response to treatment with inhibitor at

300 nM in the presence of CSPG4

1

H

2

I

30

(H) Western blot for PRAS40. PRAS40 resists deactivation in response to

treatment with inhibitor in the presence of CSPG4.

Quantitative densitometry when normalized to total protein (I) WM1341D resisted

deactivation of PRAS40 at higher doses (J) 1205LU resisted deactivation of

PRAS40 in general in response to wide range of drug treatment.

31

Discussion: The long-term prognosis for advanced stage melanoma is poor. Despite the advances in

drug discoveries, treatment resistance remains clinically a significant challenge.

Chondroitin sulfate proteoglycan is robustly expressed in melanoma cells and

associated with treatment resistance and poor prognosis. Its contribution to modulating

the BRAF pathway in response to therapy has been well studied. Our study looked at

the possible interaction of PI3k/AKT/mTOR pathway with CSPG4 and whether it

CSPG4 may instigate resistance to PI3k/AKT/mTOR pathway inhibition.

Our data shows that CSPG4 causes melanoma cells to become resistant to growth

inhibition and its knockdown sensitized them when to treatment with PF-05212384. We

also proved that CSPG4 provides protection to the inhibition of PI3k/mTOR in the

presence of serum, as we didn’t observe any difference in survival between the CSPG4

expressing and deficient cells when treated in the absence of serum with or without

IGF-1 receptor stimulation. We also observed that the metastatic cell line 1205LU

showed no difference in growth inhibition when treated with higher dose in the

presence or absence of CSPG4, however the CSPG4 deficient cells were in particular

sensitive to the lower dose of the inhibitor. Clinically, this could mean that the

metastatic disease could respond to smaller dose of inhibitor when CSPG4 is

simultaneously inhibited. We also observed a decrease in motility and invasion of the

radial growth phase and metastatic cell line when treated with the inhibitor. These cell

lines were however less sensitive to the treatment in the presence of CSPG4. Since the

rapamycin insensitive mTOR-Rictor complex is responsible for modulating the actin

32

cytoskeleton, our data indicates the efficiency of PF-5212384 in inhibiting this complex

in addition to the rapamycin sensitive mTOR-Raptor complex. These results indicate

that the inhibition of PI3k/mTOR reduces the metastatic potential of melanoma cells

and CSPG4 causes a resistance to this inhibition. We also observed an increase in

apoptosis in the absence of CSPG4 in the radial growth phase cell lines, suggesting that

a combination of this inhibitor and CSPG4 inhibition could increase apoptosis during

the early stages of the disease.

The PI3k/mTOR pathway consists of multiple signaling molecules, which interact with

each other. AKT is activated in response to various stimuli in a PI3k dependent manner.

AKT interacts with a number of molecules to regulate cell proliferation, differentiation

and survival. The activity of AKT is negatively regulated by PTEN. Proline rich AKT

substrate of 40kDa(PRAS40) is known to have a critical role in the PI3k/mTOR

pathway. PRAS40 is activated by the PI3k/AKT and binds to mTOR to mediate the

signals to it. In our metastatic cell line PRAS40 resisted complete deactivation even at

higher dose. The resistance was however more prominent the CSPG4 expressing cell

line. The vertical growth phase WM1341D cell line also resisted complete deactivation

of PRAS40 in the presence of CSPG4 indicating that CSPG4 plays a role in causing the

resistance of the PRAS40 in response to therapy in advanced stage disease. P70s6k is

downstream target of mTOR. We observed that the radial growth phase and metastatic

cell lines were less sensitive to the inhibition of this pathway in the presence of CSPG4.

4ebp1 inhibits proteins translation by binding and inactivating eIF4E. Its

phosphorylation by mTOR breaks this association, causing an increase in the cap

33

dependent protein translation. We observed a significant resistance to the

dephosphorylation of 4ebp1 in the presence of CSPG4 in all three cell lines. It has been

reported that the inhibitor of mTOR-Raptor complex rapamycin has a differential ability

to inhibit the 4ebp1 (Choo, A. Y et al 2008). This molecule has a potential to escape the

initial inhibition. Our results indicate that CSPG4 maybe causing an increase in the rate

of recovery of 4ebp1, hence affecting the cell survival.

FIGURE 9. Proposed model of resistance to PI3k/mTOR inhibition in the presence

of CSPG4.

CSPG4 provides resistance to melanoma in response to treatment with PF-5212384

at different stages of melanoma. During early growth phase CSPG4 effect on

p70s6k and 4ebp1 downstream of mTOR may provide resistance to growth and

motility inhibition. In vertical growth phase stage PRAS40 and 4ebp1 may be

meditators of CSPG4 induced resistance to growth inhibition. During metastatic

34

stage, PRAS40 and p70s6k may be involved in mechanism of CSPG4 triggered

resistance.

Image adapted from http://bit.ly/MbN04z

35

Conclusion:

Due to the better understanding of the melanoma cell biology, targeted therapies could

offer better treatment options clinically. In the recent years, a lot of interest has

developed in investigating the role of CSPG4 in melanoma cell biology. Our results

prove that PI3k/mTOR pathway is modulated by the CSPG4 and combined inhibition of

these two could result in better treatment outcome. CSPG4 causes resistance to growth

inhibition and prevents apoptosis in early stages of the disease. It also makes the cell

less responsive to the inhibition of motility and invasion. The potential mechanism of

resistance appears to be due to the interaction of CSPG4 and 4ebp1 in early and

advanced stage disease. In the vertical and metastatic disease stage PRAS40 seems to be

involved as well.

36

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