A review presentation of a paper published in British Journal of Cancer titled Cellular responses to ErbB-2 overexpression in human mammary luminal epithelial cells: comparison of mRNA and protein expression.

1. British Journal of Cancer (2004) 90,
173 – 181. doi:10.1038/sj.bjc.6601458
www.bjcancer.com
& 2004 Cancer Research UK
Cellular responses to ErbB-2
overexpression in human
mammary luminal epithelial
cells: comparison of
mRNA and protein
expression
Group 09

2. Contents
03 Methodology
01 Introduction
02 Objectives
04 Data analysis and Quantification
05 Discussion
06 Conclusion
01

3. INTRODUCTION
02

4. Introduction
What is the ErbB-2?
ErbB-2 is a member of the ErbB family of growth factor receptors comprising EGFR (ErbB-1), ErbB-2, ErbB-3
and ErbB-4.
Receptor tyrosine-protein kinase ErbB-2, also known as CD340 (cluster of differentiation 340), proto-
oncogene Neu, Erbb2 (rodent), or ErbB2 (human), is a protein that in humans is encoded by the ERBB2
gene.
ErbB is abbreviated from erythroblastic oncogene B, a gene isolated from avian genome. It is also frequently
called HER2 (from human epidermal growth factor receptor 2) or HER2/neu.
HER2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family.
Amplification or over-expression of this oncogene has been shown to play an important role in the
development and progression of certain aggressive types of breast cancer.
In recent years, the protein has become an important biomarker and target of therapy for approximately 30%
of breast cancer patients
03

5. Introduction Cont.
Function of the ErbB-2
The ErbB family consists of four plasma membrane-bound receptor tyrosine kinases. One of which is ErbB-2,
and the other members being epidermal growth factor receptor, ErbB-3 (neuregulin-binding; lacks kinase
domain), and ErbB4.
All four contain an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain
that can interact with a multitude of signaling molecules and exhibit both ligand-dependent and ligand-
independent activity.
No ligands for HER2 have yet been identified. HER2 can heterodimerise with any of the other three receptors
and is considered to be the preferred dimerization partner of the other ErbB receptors.
Dimerization results in the auto phosphorylation of tyrosine residues within the cytoplasmic domain of the
receptors and initiates a variety of signaling pathways.
04

6. Figure 1: Schematic diagram of HER2
signaling-pathways Upon ligand binding
Introduction Cont.
Signal transduction
Signaling pathways activated by HER2 include
• mitogen-activated protein kinase (MAPK)
• phosphoinositide 3-kinase (PI3K/Akt)
• phospholipase C γ
• protein kinase C (PKC)
• Signal transducer and activator of transcription (STAT)
In summary, signaling through the ErbB family of receptors
promotes cell proliferation and opposes apoptosis, and
therefore must be tightly regulated to prevent uncontrolled
cell growth from occurring.
https://www.researchgate.net/figure/Schematic-diagram-of-HER2-signaling-pathways-Upon-
ligand-binding-dimerization-between_fig2_311652882 05

7. Introduction Cont.
Clinical significance
Cancer
Amplification, also known as the over-expression of the ERBB2 gene, occurs in approximately 15-
30% of breast cancers. It is strongly associated with increased disease recurrence and a poor
prognosis; however, drug agents targeting HER2 in breast cancer have significantly positively
altered the otherwise poor-prognosis natural history of HER2-positive breast cancer.
HER2 is co-localized and most of the time, co-amplified with the gene GRB7, which is a proto-
oncogene associated with breast, testicular germ cell, gastric, and esophageal tumors.
HER2 proteins have been shown to form clusters in cell membranes that may play a role in
tumorigenesis.
06

8. Introduction Cont.
07
Main analytical techniques used in this study
1. DNA Microarray Analysis 2. 2D-Difference Gel Electrophoresis
3. Immunoblotting/Western Blotting

9. Introduction Cont.
1. DNA microarray aka DNA chips analysis
08
 DNA microarrays are solid supports, usually of glass or silicon, upon which DNA is attached
in an organized grid fashion. Each spot of DNA called a probe represents a single gene.
 The principle of DNA microarrays lies on the hybridization between the nucleotide. Using
this technology the presence of one genomic or cDNA sequence in 100000 or more
sequences can be screened in a single hybridization.
 There are 2 types of DNA microarrays;
1. cDNA based microarray
This type of chips are prepared by using cDNA.
The cDNAs are amplified by using PCR. Then,
immobilized on solid support made up of nylon
filter of a glass slide.
2. Oligonucleotide based microarray
This type of chips are prepared by series of short
(typically 20~30 bases) single-stranded DNA
sequences immobilized to a glass chip.
Arrangement of the oligos as each adjacent oligo
differs from its neighbor only at the last base.

10. Introduction Cont.
09
The basic process of a DNA microarray
a) A DNA chip can be manufactured to contain
hundreds of thousands of synthetic ssDNA
sequences.
b) Unknown DNA from a patient is separated
into single strands, enzymatically cut and
labelled with a fluorescent dye.
c) The unknown DNA is inserted into the chip
and allowed to hybridize with the DNA on
the chip.
d) The tagged DNA will bind only to the
complementary DNA on the chip. The bound
DNA will detected by its fluorescent dye and
analyzed by a computer.
https://www.researchgate.net/figure/Schematic-representation-of-the-experimental-steps-leading-to-expression-
DNA-microarray_fig1_290300802
Figure 3: Basic process of DNA
microarray

11. Introduction Cont.
10
Applications of DNA microarray
 Diagnostic and genetic engineering
 Alternative splicing detection
 Gene expression profiling
 Proteomics
 Functional genomics
 DNA sequencing
 Discovery of drugs
 Toxicological research Figure 4: A DNA microarray
image

12. Introduction Cont.
2. 2D-Difference Gel Electrophoresis
11
 2D-Difference Gel Electrophoresis (2D DIGE) is a modified form of 2D Electrophoresis
(2DE) that allows one to compare two or three protein samples simultaneously on the same
gel.
 The principle applied was very simple: proteins were resolved on a gel using isoelectric
focusing (IEF), which separates proteins in the first dimension according to their isoelectric
point, followed by electrophoresis in a second dimension in the presence of sodium dodecyl
sulfate (SDS), which separates proteins according to their molecular mass.
 The objective of separating proteins using 2D-PAGE is twofold:
(i) identifying new proteins
(ii) measuring their relative abundance between comparative samples.
 One advantage of 2D-PAGE as a separation technique is it not only resolves large numbers
of proteins, but staining these proteins enables the relative abundances of the proteins to be
quantified.

13. Introduction Cont.
2. 2D-Difference Gel Electrophoresis cont.
12
Figure 5: A schematic diagram of the 2D-DIGE
protocol
Figure 6: 2D-DIGE fluorescence images of
insecticide-treated insect Spodoptera sf-21 cells
(resistant Cy3-labeled and sensitive Cy5-labeled).
The right-hand panel is an overlay of the two
images.
https://www.future-science.com/doi/10.2144/000112823
https://link.springer.com/protocol/10.1007/978-1-61779-573-2_2

14. Introduction Cont.
3. Immunoblotting aka Western Blotting
13
 A western blot is a laboratory method used to detect specific protein molecules from among
a mixture of proteins. This mixture can include all of the proteins associated with a particular
tissue or cell type.
 Western blots can also be used to evaluate the size of a protein of interest, and to measure
the amount of protein expression.
The workflow of Immunoblotting technique
1. The first step is to prepare the protein sample by mixing it with a detergent called sodium dodecyl
sulfate (SDS), which makes the proteins unfold into linear chains and coats then with a negative charge.
2. Next, the protein molecules are separated according to their sizes using a method called gel
electrophoresis.
3. Following separation, the proteins are transferred from the gel onto a blotting membrane.
4. After the transfer is complete, the membrane carries all of the protein bands originally on the gel.

15. Figure 7: A schematic diagram of the Immunoblotting
protocol
Introduction Cont.
14
The workflow of Immunoblotting technique cont.
5. Next, the membrane goes through a treatment called
blocking, which prevents any nonspecific reactions from
occurring. The membrane is then incubated with an
antibody called the primary antibody, which specifically
binds to the protein of interest.
6. Following incubation, any unbound primary antibody
is washed away, and the membrane is incubated yet
again, but this time with a secondary antibody that
specifically recognizes and binds to the primary
antibody.
7. The secondary antibody is linked to a reporter
enzyme that produces color or light, which allows it to
be easily detected and imaged.
https://www.sciencedirect.com/topics/medicine-and-dentistry/western-blotting

16. Objectives
15

17. Objectives
16
 To gene expression profiling of both normal(HB4a) and tumor cells(C3.6)
 To validate the correlation between mRNA and protein expression of
ErbB-2-overexpressing cells
 To determine and compare the proteins expressed and their quantities in
both normal and tumor cells
 To determine the changes occurring in cell adhesion properties of normal
cells compared to that of tumor cells
 To determine the changes occurring in ErbB mediated specific mitogenic
signaling cascade of both normal and tumor cells

18. METHODOLOGY
17

19. Methodology
Cell lines, tissue culture, mitogen stimulation and RNA extraction
Generated parental HB4a mammary luminal epithelial cell line and derivatives overexpressing ErbB2
(C3.6 and C5.2)
Cells were cultured in,
RPMI-1640 media with 10% (v/v)
Foetal calf serum (FCS),
2 mM glutamine,
100 IU/ml penicillin,
100 mg/ml streptomycin,
5 mg/ml hydrocortisone
5 mg/ml insulin
at 370C in a 10% CO2-humidified incubator. 18

20. Methodology Cont.
The luminal epithelial cell line was maintained under the same growth
conditions and derived from normal human mammary luminal
epithelial cells by immortalization with SV40 large T antigen and
human telomerase reverse transcriptase.
Immortalization????
The breast tumor cell lines BT474, MCF-7, SKBr3, MDA-MB-361, MB-
435 and MB-453 were obtained from the American Type Culture
Collection (ATCC)
19

21. Methodology Cont.
For stimulation experiments, HB4a and C3.6 cells at 50 – 60% confluency were starved for 48 h
in RPMI-1640 media (serum-starvation media) with 0.1% FCS, 2 mM glutamine,
100 IU ml1 penicillin,
100 mg ml1 streptomycin and
5 mg ml1 hydrocortisone
Cells were then stimulated by the addition of 1 nM HRGb1 (R&D Systems) for 1, 4, 8, 12, 18 and 24 h.
Four replicate plates of cells were stimulated for each timepoint.
1h
1h
1h
4h
4h
4h
4h
8h
8h
8h
8h
12h
12h
12h
12h 18h
18h
18h
18h
24h
24h
24h
24h
2
1h
×
20

22. Methodology Cont.
Total RNA was isolated from each replicate experiment using TRIZOL Reagent
https://www.zymoresearch.com/pages/what-is-trizol 21

23. Methodology Cont.
Microarray experiments
Microarray analyses were performed on RNA from each of the four replicate
experiments. A ‘reciprocal duplicate’ labelling strategy was used to minimize the
effects of dye bias and to provide statistical confidence.
HB4a C3.6
A B B
C C
D D
Cy3 Cy5 Cy5 Cy5 Cy5
Two hybridizations were performed with the HB4a sample labelled with Cy3 and the C3.6 sample labelled with
Cy5 (duplicates), and two hybridizations were performed with the labelling reversed (reciprocal duplicates).
22

24. Methodology Cont.
Microarray technique
Figure 8: Dual colour fluorescence cDNA microarray analysis
workflow
https://rep.bioscientifica.com/view/journals/rep/130/1/1300001.xml 23

25. Methodology Cont.
 Probes consist of sequence-verified cDNA features spotted onto amine-binding
glass slides
 Used direct Cy dye incorporation for generation of fluorescently labelled cDNA
targets.
 25 mg of RNA was used to produce labelled cDNA by anchored oligo(dT)-primed
reverse transcription with Superscript II Reverse Transcriptase, in the presence of
Cy3- or Cy5-dUTP
 Unincorporated dye was removed using Autosequ-50 Columns and repetitive DNA
sequences were blocked with 8 mg Cot1 and 8 mg poly(dA) (Sigma) DNA before
hybridization at 470C overnight.
24

26. Methodology Cont.
Differential Protein Expression - To test the protein expression and
their levels
 The DPE data were taken by comparing the proteomes of serum-starved HB4a
and C3.6 cells using 2D-difference gel electrophoresis (2D-DIGE).
 The expression of a further 27 proteins was compared in serum-starved
HMLECs by immunoblotting with specific antibodies, using enhanced
chemiluminescence for detection, and densitometry for quantification.
 Three replicate blots were scanned and quantified for each protein, and the
average ratio (C3.6/HB4a) of C3.6 vs HB4a was determined.
- Detected statistically significant (P<0.05) expression differences for 16 proteins that
were present on the microarray.
25

27. Methodology Cont.
Cell-adhesion Assays - To test the ability of cell adherence
 Cells were trypsinised.
 Washed twice in serum-free media and seeded into 96-well plates.
 Cells were allowed to adhere for 1h at 370C, and then washed with PBS
buffer.
 A volume of 50 μl of 1 mg/ml, 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl
tetrazolium bromide (MTT) in serum-free media was added and cells were
further incubated for 4 h.
 A volume of 100 μl of DMSO was added, then plates were shaken for 20 min
at 220C, and the absorbance read at 540 nm.
- In either serum-free media, or serum-free media plus 1 nM HRGβ1.
- Wells were either untreated (adhesion to plastic) or coated with human plasma
Fibronectin (adhesion to FN).
26

28. Methodology Cont.
Media-swap experiment – Test for ErbB-specific mitogenic signaling
 The serum-starved HB4a and C3.6 cells were treated for 4h with growth
factors (EGF – 1 nM or HRGβ1 – 1nM) and generated the Conditioned
media (CM).
 Again, extensive washing and replacement with serum-free media for a
further 4h.
 The conditioned media was used to stimulate serum-starved HB4a and C3.6
cells for 15 min.
 Then, mitogenic signaling was monitored by immunoblotting with
phosphospecific antibodies to ERK1 and 2 (Extracellular signal-regulated
kinases).
- CM media was added to serum-starved cells that had been pretreated with 5 mM
AG-1478 (a potent ErbB-kinase inhibitor) for 30 min.
27

29. DATA ANALYSIS
&
QUANTIFICATION
28

30. Data analysis and Quantification
 Microarrays were scanned using a ScanArray 4000XL (Packard BioChip
Technology, Billerica, MA, USA)
 The expression level of each probe was derived by dividing the fluorescence
intensity of the C3.6 result by the HB4a result for each probe in the four replicates.
 Immunoblotting assays and 2D-DIGE, detection using CCD camera imaging
devices and quantification using densitometry were done. (Bio-Rad &
QuantityOne)
- The raw image data were acquired using ScanArray v3.0 software, and then
exported to QuantArray v3.0 software for data extraction.
- Normalizing of signal intensities of the Cy3 and Cy5 channels extracted data then
loaded into GeneSpring v4.2 (Silicon Genetics, Redwood City, CA, USA).
- When the control channel value was below 10.0, the data point was considered bad.
- The final ratio is the geometric mean of the four replicates.
- Expression differences were considered significant if the ratio of C3.6 : HB4a was
≤0.5 (downregulated), or ≥2.0 (upregulated).
29

31. RESULTS
30

32. Results
Global changes in gene expression
ErbB-2
overexpressing
C3.6 cells and
their normal
counter parts
HB4a serum
starved and treat
with HRGβ1
Figure 9: Analysis of global differences in mRNA
expression between C3.6 and HB4a Human mammary
luminal epithelium cells (HMLECs)
31

33. Results Cont.
Identification of ERBb-2-dependent mRNA changes
 Two cell lines compare by co-hybridizing labelled cDNA.
Heregulin b1 stimulation.
 A 9932 number of probes present on the microarray, 667 probes displayed a
greater than two-fold difference in expression between C3.6 and HB4a cells.
Number of gene with different expression not differ between T1 and T12
in time point T0, T18, T24 has significant different.
C3.6 high growth factor
C3.6 proliferative capacity
Response to HRGβ1
32

34. Results Cont.
Identification of ERBb-2-dependent mRNA changes cont.
Gene constitutively
high/low
Expression effected directly
to ErbB-2 overexpression
 21 genes upregulated (including ErbB-2)
 27 genes downregulated
 1/3 genes are upregulated
 Upregulation occur secondary metabolic process
 ErbB-2 overexpressing  increased cyclic dependent kinase activity
 Downregulation of ISGF3 gamma subunit with STAT1 and STAT2
 ISGF3 complex  IFNα/β gene transcription
33

35. Results Cont.
Correlation between gene expression and protein expression
Figure 10: 2D-DIGE analysis resultant images (A) Upregulation of ErbB-2, carbamoyl
phosphate synthetase 1 (CPS1I), Copine III, ubiquitin C-terminal hydrolase L1 (UCHL1)
and cyclin D2 (CCND2) protein in C3.6 and C5.2 ErbB-2-overexpressing HMLECs.
(B) Protein expression (by immunoblotting) of ErbB-2, EGFR, ErbB-3, CPS1, Copine III,
ISGF3G and MxA in a panel of breast tumor cell lines.
34

36. Results Cont.
Correlation between gene expression and protein expression cont.
ERM protein radixin
(RDX)
CPS 1
LCP 1
HIBCH
Copine Ⅲ
UCHC Ⅰ
 HB4a control cell line
 C3.6 and C5.2 cell line experimental cell line
 Variety of proteins overexpress in tumor cell lines
 C5.2 protein expression level is higher than C3.6 cell line
Upregulated proteins identified through 2D-DIGE analysis.
35

37. Results Cont.
Correlation between gene expression and protein expression cont.
Figure 11: The correlation between
mRNA and protein ratios of 43 genes in
serum-starved C3.6 cells, relative to
HB4a cells.
 mRNA data derived from microarray results.
 Comparison of the protein and mRNA ratios for 43 genes
showed a statistically significant and high correlation
coefficient of 0.81 of 0.66.
 mRNA expression correlates strongly and positively with
increased translation and protein expression.
36

38. Results Cont.
ErbB-2 dependent modification of HMLEC adhesion
Figure 12: Cell adhesion assays on C3.6 and
HB4a in serum-free media, or media
supplemented with 1mM HRGb1 as measured
by incorporation of MTT into living cells (A540
nm).
 Degradation and cell adhesion were also found to be
differentially expressed following HRGb1 treatment.
 Serum-starved C3.6 cells were significantly less
adhesive than HB4a cells on plastic.
 FN-coated plastic and HRGb1 treated one increased
adhesive in C3.6 cells.
37

39. Results Cont.
Differential regulation of autocrine/paracrine signaling by ErbB-2
Figure 13: (A) Expression profile (C3.6 : HB4a) of
amphiregulin precursor (AREG) across the time course of
HRGb1 stimulation. (B) Treatment of serum-starved cells
with conditioned media
from EGF or HRGb1-stimulated cells results in an ErbB
dependent activation of MAPK signaling.
38

40. Results Cont.
Differential regulation of autocrine/paracrine signaling by ErbB-2 cont.
Figure 14: (C) HRGb1-induced
expression profile of Fos and Jun
family members (components of the
AP-1 transcription complex) in C3.6 vs
HB4a cells.
 Used to stimulate serum starved and AG-1478-treated
HB4a and C3.6 cells for 15 min.
 Heregulin1 stimulation hours increase Normalized
intensity C3.6 : HB4a up and down in AP-1
transcription complexes.
39

41. DISCUSSION
40

42. Discussion
Parallel microarray and proteomic analysis were conducted to
investigate ErbB-2- and HRGβ1- dependent changed in protein
expression in model cell systems of breast cancer.
By using different cell types, quantification methods and different
sets of genes.
The correlation between mRNA and
protein expression for 43 genes were
very high
Previous studies using
human liver, lung
adenocarcinomas show
lesser correlation
41

43. Discussion cont.
They are,
genes with roles in cellular metabolism
regulation of cell cycle progression
IFN signaling
cell adhesion
autocrine/paracrine signaling
AP-1- mediated transcription
Expression of the majority of genes is controlled at the level of
transcription in response to ErbB-2 overexpression and a minor
number of genes were regulated post-transcriptionally.
Six functional groups of genes and proteins were identified with
altered expression.
42

44. A recent study by Mackay et al., 2003 also reported microarray
analysis of genes associated with ErbB-2 expression in the HB4a,
C3.6, C5.2 model system
Number of differentially expressed genes identified in
the two studies are the same:
Constitutively Up regulated - S100P, CPS1, vimentin
constitutively down regulated - IGFBP3, FN1, SPARC
 12 other genes differentially expressed
 Several novel gene expression changes –
CCND2, ISGF3G, UCHL1, LCP1 and CPNE3
Discussion cont.
43

45.  Affect of cdk activity for proliferation of HMLEC
 Due to increased cyclin D1 and cdk6 protein expression and
reduction in the expression and binding of the cdk inhibitor p27 to
cdk2.
In the ErbB-2-overexpressing cells, enhanced MAPK activation played a critical role in cell cycle
progression and proliferation by promoting cdk6 and cdk2 activities.
This study shows the effect of HRGb1-dependent
downregulation of three cdk inhibitors and the
constitutive upregulation of cyclin D2 in ErbB-2-
overexpressing cells
By a previous study,
Discussion cont.
44

46. Discussion cont.
ultimately
Down regulation of cdk
inhibitors
Promote cell cycle
progression through
activation of cdk2
Constitutive upregulation
of CCND2
Reduce the
dependance of
proliferation of breast
cancer cells on
serum
Cell cycle progression of ErbB-2-dependent cells and
increased tumorigenic potential in ErbB-2-
overexpressing luminal cells.
45

47. Discussion cont.
 The effect IFN signaling and adherence for increased proliferative
capacity of C3.6 cells
 IFNs are anti-proliferative and can promote apoptosis under certain
conditions.
In the ErbB-2-over expressing cells multiple IFN-inducible genes, mRNA and proteins were down
regulated.
Strong inverse correlation between ErbB-2 expression and ISGF3G expression
ISGF3G - insulin-like growth factor-binding protein 3
critical component of ISGF3 transcriptional activator complex for many IFN-inducible genes
46

48. Discussion cont.
 The effect of HRGb1 treatment for anchorage dependant cellular
adhesion
 Ex:- FN1, TIMP3 and FBLN2 were all constitutively down-regulated
by affecting integrin mediated cellular adhesion.
After HRGb1 treatment differential expression was observed in a significant number of ECM
components or genes involved in ECM modelling, degradation and cell adhesion
ErbB-2 suppression of FN1 plays a
functional role in anchorage-
independent growth and metastasis
By a previous study, By this study,
HRGb1 can modify adhesion, and
enhance the invasiveness
47

49. Discussion cont.
 Increased temporal MAPK signaling in ErbB-2 overexpressing
cells
 First phase is induced by HRGb1
Second phase which is mediated via MAPK signaling is induced
by autocrine/paracrine growth factor production
Temporal differential expression of AP-1 transcription complex components after
stimulation with HRGb1
Suggestive of biphasic transcriptional activity
48

50. Discussion cont.
In the cells treated with EGF or HRGb1 activation of MAPK signaling was observed
Activation could be blocked by inhibition of ErbB receptor kinase activity
Therefore, it can be concluded that autocrine signaling which contribute to
enhanced proliferation is ErbB-dependent
49

51. CONCLUSION
50

52. Conclusion
ErbB-2 overexpression induces
 increased temporal MAPK signaling
increased cdk activity (via up-regulation of cyclin D1 and CCND2,
and down-regulation of cdk inhibitors)
inhibition of basal IFN signaling and reduced cellular adhesion
Anchorage-independent proliferation of HMLECs to promote tumorigenesis
51

53. Group Members
1. Eranga Mudalige
2. Mohamed Rifath
3. Sachintha Rathnayake
4. Mohamed Jeleel
5. Hashini Madumali
6. Heshan Gamage
52

54. THANK YOU!

55. QUESTIONS?
ANY