Aveneu Park, Starling, Australia

Background: clinical data sets. PCa cells LNCaP,

Background: cap-dependent translation is necessary due to high
protein requirement in cancer. An interaction between EIF4E and EIF4G is
crucial for EIF4F complex formation and initiation of cap-dependent
translation. In the present study, we analyzed Human prostate cancer
tissue microarray(TMA) and mRNA data for EIF4G1 in clinical datasets, and prostate
tumor tissue from TRAMP(Transgenic Adenocarcinoma of Mouse Prostate) model. We
also assessed the functional role of EIF4G1 in commonly used PCa cell lines.

Methods: TMA was used to analyze the EIF4G1 protein levels in
patient samples and mRNA data for EIF4G1 was analyzed from TCGA and
Trento/Cornell/Broad clinical data sets. PCa cells LNCaP, C4-2b, 22Rv1, DU145,
PC3 and human prostate cells RWPE-1 were used. For an in-vivo model of PCa, we used TRAMP and wild-type mouse. Loss of
function studies was performed by using siRNA/shRNA. Real-time(RT) PCR and
Western Blot analysis were used to quantitate relative mRNA and protein levels
respectively. Analysis of polysome was performed by sucrose density gradient
fractionation and Polysome-to-Monosome(P/M) ratios were determined. Cell cycle,
cell proliferation/migration, and Clonogenic activity were measured by standard

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Results: TMA analysis showed that protein levels of EIF4G1 are
high in PCa as compared to normal prostate tissue, and there is a graded
increase in EIF4G1 as the disease progresses. TCGA dataset revealed that EIF4G1
positively correlated with higher tumor grade and stages and Trento/Cornell/Broad
dataset showed that 43% of castration-resistant prostate cancer(CRPC) patients
have EIF4G1 mRNA up-regulation. PCa cells express a significantly higher level
of EIF4G1 as compared to normal prostate cells. Similarly, prostate tumor
tissue from TRAMP tissue showed higher EIF4G1 expression as compared to normal
wild-type prostate tissue. There is a shift in P/M ratio with the siEIF4G1
knockdown. Silencing of EIF4G1 causes decreases in Cyclin D1 and p-Rb levels and
G0/G1 cell cycle delay and impaired Clonogenic activity as well as cell
proliferation. RT-PCR data suggest that EIF4G1 knockdown decreases the level of
EMT markers and limits the cell migration.

Conclusions: Taken all together, our data indicate that EIF4G1 may
function as an oncoprotein and is a novel target for intervention in PCa and

Keywords: EIF4G1, Prostate Cancer, Tissue Microarray, TRAMP, Polysome












161,360 new prostate cancer cases has been predicted for this year 1. Current
treatment for localized tumor is surgery or chemotherapy and for metastases
tumor, the primary target is Androgen receptor, which is the key molecular driver
of the disease. Despite different treatment options disease is advanced and
thus further giving rise a different phenotype of the disease i.e.
castration-resistant prostate cancer in due course of time, which is
unmanageable by current therapy and leads to estimated 26,730 deaths in 2017
1. Thus, new treatment options are highly warranted for better prognosis of
the disease.

control is critical for any cancer cell growth and progression, which requires
a high protein synthesis levels and translation of specific mRNAs that are
responsible for different tumorigenic properties. Cap-dependent translation is
essential to maintain high protein synthesis in rapidly dividing cancer cells.
Translational control occurs predominately during a  rate-limiting, initiation step which is
subjected to extensive regulation 2,3 and is governed by cap-binding complex,
eukaryotic initiation factor 4F (eIF4F) which is composed of cap-binding
protein eIF4E, eIF4A (helicase) and eIF4G (scaffolding protein). In the normal
condition of cell EIF4E is constrained 4E-BPs and thus inhibits its binding with
eIF4G, thus inhibiting the eIF4F translation initiation complex formation 5. An
interaction between eIF4E and eIF4G is crucial for the formation of the eIF4F
complex and initiation of cap-dependent translation 4. EIF4G family comprises
of three isoforms eIF4G1, eIF4G2 and eIF4G3 6,7 among which eIF4GI is the
major isoform (>85%) 8. Isoforms eIF4G11, eIF4G2 and eIF4G3 have 50%
identity and their genes have been mapped to 3q27.1, 11p15 and 1p36.12
respectively 7. Isoforms eIF4G1 and eIF4G3 are involved in cap-dependent
translation, while eIF4G2 is associated with IRES-dependent translation in
cells 6,9.

studies have shown that eukaryotic translation initiation factor 4 gamma 1
(EIF4G1) is overexpressed in different cancers such as breast cancer, lung
cancer, nasopharyngeal cancer, cervical cancer, multiple myeloma and ovarian
cancer 10-17 and related to tumorigenesis and pathogenesis 13. It’s known that
interaction of EIF4G1-EIF4E not only govern the protein synthesis but also its
quality and thus contribute to the cell phenotype and function. 18. Chromosomal
location of eIF4G1 (3q27.1) is known to amplified in PCa patients 19.

recent studies have demonstrated that EIF4G1 plays important roles in different
cancers, its functional role in Prostate Cancer (PCa) is unreported. We wanted
to explore the functional role of EIF4G1 in PCa cells. We focused on the role
of EIF4G1 in the disease progression, translation control, cell cycle
distribution and cell characteristics such as growth and proliferation and
migration in PCa.

Materials and


All chemicals were purchased from Sigma-Aldrich.
Antibodies for EIF4G1 (Cell signaling #2498); Cyclin D1 (Santa Cruz sc-20044);
pRb (Cell signaling #9308); ?-tubulin (Developmental Studies Hybridoma Bank
E7); ?-actin (Sigma A2228) were used to probe respective proteins of interest.

Human Prostate
tissue Microarray & Immunohistochemistry (IHC):

Tissue Microarray for prostate (US Biomax, Inc. #PR1921a)
was used to see the EIF4G1 protein levels in the patient sample. This TMA
contains 80 cases of adenocarcinoma, 8 adjacent normal prostate tissues and, 8
normal prostate tissues. Following lab protocol antigen-retrieval was done,
followed by blocking in 3% BSA and incubated with EIF4G1 antibody overnight in the
humidified chamber at 4°C. Further tissue section was incubated with the biotinylated
secondary antibody, followed by DAB staining and counterstained with
hematoxylin (Sigma-Aldrich). Images for representative tissue sections were
taken by Olympus BX51 microscope at the magnification of X20 and X40 using
CellSens Entry. Above IHC protocol was also used for EIF4G1 staining with
wild-type as well as 30-week old TRAMP prostate tissue.

TCGA data mining
and retrieval of data from the clinical data set:

mRNA expression and clinical data from TCGA (The
Cancer Genome Atlas) data set for the prostate cancer and normal samples were
analyzed by UALCAN (http://ualcan.path.uab.edu/) 20 web server and TCGA
database. The analysis was done on 497 primary tumors of prostate cancer and 52
normal samples from TCGA. Using a gene name (or more), the web server will mine
the data available for the gene expression with cancer stage, Gleason score and
survival analysis and statistical significant p-value for each group/subgroup
analysis. To study gene expression changes from different clinical dataset we
used freely accessible cBioPortal (http://www.cbioportal.org) tool 21, 22.
All prostate tumors with mRNA expression data (n=114) from the Neuroendocrine
Prostate Cancer dataset 23  using a mRNA Z-score
threshold of ± 2 as compared with normal prostate samples was used for EIF4G1.
Genetic alterations in percent mRNA upregulation were taken consideration for the
present study.

Cell Line/Culture
& Tissue samples:

Prostate cancer cell lines
LNCaP, C4-2b, 22Rv1, DU145 were cultured in RPMI 1640 complete medium (Hyclone:
Cat No.:30255.01) and PC3 cells were cultured in DMEM/F12 with 10% v/v Fetal
Bovine Serum, 1% v/v Antibiotics (Penicillin and Streptomycin). The normal
Prostate cell line RWPE-1 was cultured in Keratinocyte-SFM (ThermoFisher Cat
No.: 17005042) with EGF 1-53 (Epidermal Growth Factor 1-53) and BPE (Bovine
Pituitary Extract). The cells were incubated at 37°C in a 5% CO2 humidified
atmosphere. Further Prostate tumor tissue of 30 Week old Transgenic
Adenocarcinoma of the Mouse Prostate (TRAMP) mice and prostate tissue from
wild-type mice were used in present study.


Polysome-bound RNA
fractionation was done by the method described somewhere else 24 with modifications. 10×106 cells for
LNCaP and C4-2b siControl and siEIF4G1 cells were used per sucrose
gradient. Briefly, before harvesting, cells were pulsed with 100 µg/ml of Cycloheximide
for 10 minutes. And lysed in PL Buffer (Polysome lysis) containing 20 mmol/L
Tris-HCl (pH 7.5), 250 mmol/L NaCl, 15 mmol/L MgCl2, 0.5% NP-40, 100 µg/mL Cycloheximide,
2 mmol/L DTT, 50 µg/mL heparin, and 200 U/mL RNasin (Promega) and homogenized.
Lysing 15 minutes on ice, cell lysates were centrifuged and the supernatant was
loaded onto a 10% to 60% sucrose gradient tube. Tubes were centrifuged at
35,000g for 3 hours at 4°C and fractions were collected using Density Gradient
Fractionation System by ISCO with continuous monitoring based on an absorbance
at 254nm. To calculate polysome to monosome ratio graph were scanned and pixels
of polysome and monosome were measured with tpsUtil64 and
tpsDIG2w64 software. (http://life.bio.sunysb.edu/morph/).




Immunoblotting for EIF4G1 Protein
expression was done in above PCa cell lines as well as in normal prostate
tissue and in TRAMP prostate tumor tissue. Further immunoblotting for EIF4G1/pRb/CyclinD1
was done on siControl and siEIF4G1 in LNCaP & C4-2b cells. Blot was scanned
for the respective protein of interest and loading control protein by LI-COR
Odyssey CLx (LI-COR, Lincoln, USA) system by using IRDye 680 goat anti-mouse
and IRDye 800 goat anti-rabbit secondary antibodies or HRP conjugated secondary
mouse and rabbit and developed by the film.

siRNA/shRNA Mediated EIF4G1 knockdown:

For the loss of function
studies, we knockdown EIF4G1 in cells, we used siRNA for EIF4G1 (human) from
Santa Cruz (sc-35286) and non-targeting siRNA-A as a negative control from
Santa Cruz (sc-37007) And we also used shRNA vector control (SHC001) and shRNA for EIF4G1 (SHCLNG-NM_182917, TRCN0000061770) from Sigma-Aldrich. Transfection
of this siRNA/shRNA was performed in six-well plates using the HiPerFect
transfection reagent (Qiagen, CA)/Lipofectamine® 200 reagents (Life
Technologies, Invitrogen) respectively as per manufacturer’s protocol. Effect
of knockdown was checked by immunoblotting.

Flow cytometry:

Cell Cycle Analysis was
done for siControl and siEIF4G1 in LNCaP and C4-2b cells as mentioned earlier 25. Cell cycle
distributions were analyzed by using BD LSR II Flow Cytometer and ModFit LT
Software was used for analysis.


Total RNA was extracted
from siControl and siEIF4G1 in LNCaP and C4-2b cells using a commercially available RNA isolation kit (OMEGA), followed by
cDNA synthesis from 1µg of RNA. Further RT-PCR was done for EIF4G1/CyclinD1/EMT
related genes such as N-Cadherin/Vimentin/Snail and Zeb 1.

Cell proliferation
and viability:

Cell proliferation and
viability assays were done on LNCaP& C4-2b siControl and siEIF4G1 for
24/48/72/96 h by MTT assay and Crystal Violet stating respectively as described
previously 25.


LNCaP and C4-2b cell transfected with siEIF4G1 or siControl (500 cells/well) were seeded in
6-well plates. After 14 days, the cells were stained with 0.4% Crystal violet
solution for 1hr at room temperature, wash and dry the plates and visible
colonies were counted by ImageJ software.

Cell Migration Assay:

Trans-well cell migration assay was done for vector
control and shEIF4G1 in LNCaP and C4-2b cells as mentioned earlier with some
modifications 26. Briefly, a single cell suspension of VC and shEIF4G1 for
LNCaP and C4-2b cells were seeded on 8-?m-pore size insert in a Transwell. A
total of 2×105 cells in 300 µl of 1% serum medium were seeded and
media containing 20% FBS (600 ?l) was added to the lower chamber. After 24 hr
of migration time, transwell was stained with 0.4% crystal violet. The upper
side of the transwell chamber was cleaned with cotton before drying and Images of
migratory cells on the underside of the transwell were captured using above
mentioned microscope at 10X magnification. The migratory cell was counted by
counting four fields per stained membrane.

Statistical analysis:

are expressed as means ± standard deviations (SD). The two-tailed Student
t-test and ANOVA test were used for statistical analysis of experiments and
GraphPad Prism5 was used for statistical analysis. Significant differences in
p-values are indicated as *


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