© Sehrish Javaid 2012 - University of Minnesota
Post on 02-Mar-2022
5 Views
Preview:
Transcript
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.
ii
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.
iii
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.
iv
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
9
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:
10
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.
14
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:
18
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
Bibliography
Amaravadi, R. K. and K. T. Flaherty. 2007. "Targeted Therapy for Metastatic Melanoma." Clinical Advances in Hematology & Oncology : H&O 5 (5): 386-394.
Arkenau, H-T, R. Kefford, and G. V. Long. 2011. "Targeting BRAF for Patients with Melanoma." British Journal of Cancer 104 (3): 392-398. doi:10.1038/sj.bjc.6606030.
Avils, Jos and Pablo Lzaro. 2006. "[Genetic Predisposition in Cutaneous Melanoma]." Actas Dermo-Sifiliográficas 97 (4): 229-240. doi:10.1016/S0001-7310(06)73390-7.
Bauer, Jrgen, and ClausGarbe. "Acquired melanocytic nevi as risk factor for melanoma development. A comprehensive review of epidemiological data." Pigment cell research 16.3 (2003):297-306.
Bichakjian, C. K., A. C. Halpern, T. M. Johnson, A. Foote Hood, J. M. Grichnik, S. M. Swetter, H. Tsao, et al. 2011. "Guidelines of Care for the Management of Primary Cutaneous Melanoma. American Academy of Dermatology." Journal of the American Academy of Dermatology 65 (5): 1032-1047. doi:10.1016/j.jaad.2011.04.031.
Brachmann, Saskia, Christine Fritsch, Saveur-Michel Maira, and Carlos Garca-Echeverra. 2009. "PI3K and mTOR Inhibitors: A New Generation of Targeted Anticancer Agents." Current Opinion in Cell Biology 21 (2): 194-198. doi:10.1016/j.ceb.2008.12.011.
Campoli, Michael, Soldano Ferrone, and Xinhui Wang. 2010. "Functional and Clinical Relevance of Chondroitin Sulfate Proteoglycan 4." Adv Cancer Res 109: 73-121. doi:10.1016/B978-0-12-380890-5.00003-X.
Cheng, Jin, Craig Lindsley, George Cheng, Hua Yang, and Santo Nicosia. 2005. "The Akt/PKB Pathway: Molecular Target for Cancer Drug Discovery." Oncogene 24 (50): 7482-7492. doi:10.1038/sj.onc.1209088.
Choo, A. Y., S. O. Yoon, S. G. Kim, P. P. Roux, and J. Blenis. 2008. "Rapamycin Differentially Inhibits S6Ks and 4E-BP1 to Mediate Cell-Type-Specific Repression of mRNA Translation." Proceedings of the National Academy of Sciences of the United States of America 105 (45): 17414-17419. doi:10.1073/pnas.0809136105.
Courtney, Kevin, Ryan Corcoran, and Jeffrey Engelman. 2010. "The PI3K Pathway as Drug Target in Human Cancer." Journal of Clinical Oncology 28 (6): 1075-1083. doi:10.1200/JCO.2009.25.3641.
37
Davies, M. A. 2012. "The Role of the PI3K-AKT Pathway in Melanoma." Cancer Journal (Sudbury, Mass.) 18 (2): 142-147. doi:10.1097/PPO.0b013e31824d448c.
Davies, Michael. 2012. "The Role of the PI3K-AKT Pathway in Melanoma." The Cancer Journal 18 (2): 142-147. doi:10.1097/PPO.0b013e31824d448c.
Davies, Michael, Patricia Fox, Nicholas Papadopoulos, Agop Bedikian, Wen-Jen Hwu, Alexander Lazar, Victor Prieto, et al. 2012. "Phase I Study of the Combination of Sorafenib and Temsirolimus in Patients with Metastatic Melanoma." Clinical Cancer Research 18 (4): 1120-1128. doi:10.1158/1078-0432.CCR-11-2436.
Dhomen, Nathalie and Richard Marais. 2009. "BRAF Signaling and Targeted Therapies in Melanoma." Hematology/oncology Clinics of North America 23 (3): 529-45, ix. doi:10.1016/j.hoc.2009.04.001.
Eggermont, A. M. 2010. "Advances in Systemic Treatment of Melanoma." Annals of Oncology : Official Journal of the European Society for Medical Oncology / ESMO 21 Suppl 7: vii339-44. doi:10.1093/annonc/mdq364.
Flaherty, Keith T, et al. "Inhibition of mutated, activated BRAF in metastatic melanoma." The New England journal of medicine 363.9 (2010):809-819.
Garbe, C., T. K. Eigentler, U. Keilholz, A. Hauschild, and J. M. Kirkwood. 2011. "Systematic Review of Medical Treatment in Melanoma: Current Status and Future Prospects." The Oncologist 16 (1): 5-24. doi:10.1634/theoncologist.2010-0190.
Grossman, D. and D. C. Altieri. 2001. "Drug Resistance in Melanoma: Mechanisms, Apoptosis, and New Potential Therapeutic Targets." Cancer and Metastasis Reviews 20 (1-2): 3-11. doi:10.1023/A:1013123532723.
Haluska, Frank, Trevor Pemberton, Nageatte Ibrahim, and Kevin Kalinsky. 2007. "The RTK/RAS/BRAF/PI3K Pathways in Melanoma: Biology, Small Molecule Inhibitors, and Potential Applications." Seminars in Oncology 34 (6): 546-554. doi:10.1053/j.seminoncol.2007.09.011.
Huang, Jingxiang and Brendan Manning. 2009. "A Complex Interplay between Akt, TSC2 and the Two mTOR Complexes." Biochemical Society Transactions 37 (1): 217-222. doi:10.1042/BST0370217.
Lens, M. B. and M. Dawes. 2004. "Global Perspectives of Contemporary Epidemiological Trends of Cutaneous Malignant Melanoma." The British Journal of Dermatology 150 (2): 179-185.
Mallon, Robert, Larry Feldberg, Judy Lucas, Inder Chaudhary, Christoph Dehnhardt, Efren Santos, Zecheng Chen, et al. 2011. "Antitumor Efficacy of PKI-587, a
38
Highly Potent Dual PI3K/mTOR Kinase Inhibitor." Clinical Cancer Research 17 (10): 3193-3203. doi:10.1158/1078-0432.CCR-10-1694.
Mazzoletti, Marco, Francesca Bortolin, Laura Brunelli, Roberta Pastorelli, Silvana Di Giandomenico, Eugenio Erba, Paolo Ubezio, and Massimo Broggini. 2011. "Combination of PI3K/mTOR Inhibitors: Antitumor Activity and Molecular Correlates." Cancer Research 71 (13): 4573-4584. doi:10.1158/0008-5472.CAN-10-4322.
Meyle, K. D. and P. Guldberg. 2009. "Genetic Risk Factors for Melanoma." Human Genetics 126 (4): 499-510. doi:10.1007/s00439-009-0715-9.
Oliveria, S. A., M. Saraiya, A. C. Geller, M. K. Heneghan, and C. Jorgensen. 2006. "Sun Exposure and Risk of Melanoma." Archives of Disease in Childhood 91 (2): 131-138. doi:10.1136/adc.2005.086918.
Paraiso, K. H., I. V. Fedorenko, L. P. Cantini, A. C. Munko, M. Hall, V. K. Sondak, J. L. Messina, K. T. Flaherty, and K. S. Smalley. 2010. "Recovery of Phospho-ERK Activity Allows Melanoma Cells to Escape from BRAF Inhibitor Therapy." British Journal of Cancer 102 (12): 1724-1730. doi:10.1038/sj.bjc.6605714.
Paraiso, Kim H. T., Yun Xiang, Vito Rebecca, Ethan Abel, Y. A. Chen, A. C. Munko, Elizabeth Wood, et al. 2011. "PTEN Loss Confers BRAF Inhibitor Resistance to Melanoma Cells through the Suppression of BIM Expression." Cancer Research 71 (7): 2750-2760. doi:10.1158/0008-5472.CAN-10-2954.
Pollak, Michael. "The insulin and insulin-like growth factor receptor family in neoplasia: an update." Nature Reviews. Cancer 12.3 (2012):159-169.
Ppulo, Helena, Jos Lopes, and Paula Soares. 2012. "The mTOR Signalling Pathway in Human Cancer." International Journal of Molecular Sciences 13 (2): 1886-1918. doi:10.3390/ijms13021886.
Price, M. A., L. E. Colvin Wanshura, J. Yang, J. Carlson, B. Xiang, G. Li, S. Ferrone, A. Z. Dudek, E. A. Turley, and J. B. McCarthy. 2011. "CSPG4, a Potential Therapeutic Target, Facilitates Malignant Progression of Melanoma." Pigment Cell & Melanoma Research 24 (6): 1148-1157. doi:10.1111/j.1755-148X.2011.00929.x; 10.1111/j.1755-148X.2011.00929.x.
Siegel Rebecca, Naishadham Deepa, Ahmedin Jemal DVM, PhD Cancer statistics, 2012 CA: A Cancer Journal for Clinicians, Vol62, Issue 1,p 10-29
Shaw, Leslie. 2005. "Tumor Cell Invasion Assays." Methods in Molecular Biology 294: 97-105.
39
Sinnberg, T., K. Lasithiotakis, H. Niessner, B. Schittek, K. T. Flaherty, D. Kulms, E. Maczey, et al. 2009. "Inhibition of PI3K-AKT-mTOR Signaling Sensitizes Melanoma Cells to Cisplatin and Temozolomide." The Journal of Investigative Dermatology 129 (6): 1500-1515. doi:10.1038/jid.2008.379.
Venkatesan, Aranapakam, Christoph Dehnhardt, Efren Delos Santos, Zecheng Chen, Osvaldo Dos Santos, Semiramis Ayral Kaloustian, Gulnaz Khafizova, et al. 2010. "Bis(Morpholino-1,3,5-Triazine) Derivatives: Potent Adenosine 5'-Triphosphate Competitive Phosphatidylinositol-3-kinase/mammalian Target of Rapamycin Inhibitors: Discovery of Compound 26 (PKI-587), a Highly Efficacious Dual Inhibitor." Journal of Medicinal Chemistry 53 (6): 2636-2645. doi:10.1021/jm901830p.
Wang, Haitao, Qishan Zhang, Qiang Wen, Yongxin Zheng, Lazarovici Philip, Hao Jiang, Jun Lin, and WenHua Zheng. 2012. "Proline-Rich Akt Substrate of 40kDa (PRAS40): A Novel Downstream Target of PI3k/Akt Signaling Pathway." Cellular Signalling 24 (1): 17-24. doi:10.1016/j.cellsig.2011.08.010.
Wang, Jian, Agnete Svendsen, Justyna Kmiecik, Heike Immervoll, Kai Skaftnesmo, Jess Planagum, Rolf Reed, et al. 2011. "Targeting the NG2/CSPG4 Proteoglycan Retards Tumour Growth and Angiogenesis in Preclinical Models of GBM and Melanoma." PLoS ONE 6 (7): e23062-e23062. doi:10.1371/journal.pone.0023062.
Wang, Xinhui, Takuya Osada, Yangyang Wang, Ling Yu, Koichi Sakakura, Akihiro Katayama, James McCarthy, et al. 2010. "CSPG4 Protein as a New Target for the Antibody-Based Immunotherapy of Triple-Negative Breast Cancer." Journal of the National Cancer Institute 102 (19): 1496-1512. doi:10.1093/jnci/djq343.
Willems, Lise, Jerome Tamburini, Nicolas Chapuis, Catherine Lacombe, Patrick Mayeux, and Didier Bouscary. 2012. "PI3K and mTOR Signaling Pathways in Cancer: New Data on Targeted Therapies." Current Oncology Reports 14 (2): 129-138. doi:10.1007/s11912-012-0227-y.
Wu, P. and Y-Z Hu. 2010. "PI3K/Akt/mTOR Pathway Inhibitors in Cancer: A Perspective on Clinical Progress." Current Medicinal Chemistry 17 (35): 4326-4341. doi:10.2174/092986710793361234.
Yajima, Ichiro, Mayuko Kumasaka, Nguyen Thang, Yuji Goto, Kozue Takeda, Osamu Yamanoshita, Machiko Iida, et al. 2012. "RAS/RAF/MEK/ERK and PI3K/PTEN/AKT Signaling in Malignant Melanoma Progression and Therapy." Dermatology Research and Practice 2012: 354191-354191. doi:10.1155/2012/354191.
Yang, J., M. A. Price, C. L. Neudauer, C. Wilson, S. Ferrone, H. Xia, J. Iida, M. A. Simpson, and J. B. McCarthy. 2004. "Melanoma Chondroitin Sulfate Proteoglycan
40
Enhances FAK and ERK Activation by Distinct Mechanisms." The Journal of Cell Biology 165 (6): 881-891. doi:10.1083/jcb.200403174.
Yu, L., E. Favoino, Y. Wang, Y. Ma, X. Deng, and X. Wang. 2011. "The CSPG4-Specific Monoclonal Antibody Enhances and Prolongs the Effects of the BRAF Inhibitor in Melanoma Cells." Immunologic Research 50 (2-3): 294-302. doi:10.1007/s12026-011-8232-z.
Zhou, Hongyu and Shile Huang. 2011. "Role of mTOR Signaling in Tumor Cell Motility, Invasion and Metastasis." Current Protein Peptide Science 12 (1): 30-42.
Zito, Christopher R, and Harriet MKluger. "Immunotherapy for metastatic melanoma." Journal of cellular biochemistry 113.3 (2012):725-734.
top related