Understand the signaling pathways related to HER2+ breast cancer.

1. Molecular basis of HER2+
breast cancer, significance of
HER-2 and its associated
signalling pathways
Praveen Nilwala, Dihansa Hettiarachchi,
Rasanie Goonewardane, Seshani Maria
and Sureshni Fernando

2. Contents
• Introduction
• Risk factors
• HER 2 receptor
• MAPK pathway
• PI3K/AKT/mTOR pathway
• Pathogenesis
• Diagnostic tests

3. Introduction
• Breast cancer is the most commonly diagnosed cancer in females.
• Human epidermal growth factor receptor 2 (HER2) positive breast cancer
accounts for 20-30% of all breast cancers (Engel and Kaklamani, 2007).
Figure 1: Normal cell versus HER2 positive
breast cancer cell (Eldridge, 2020).
HER2
• Normally helps breast cell
growth
• When overexpressed
- tumorigenesis
Features
• More aggressive
• Poor prognosis
• Resistance to
chemotherapy
• Shorter survival rate
• High incidence of relapse
(Lv et al., 2016).

4. Risk factors
• Gender
• Age
• Late child-bearing: after 35 years of age
• Caucasian race
• Lifestyle factors
– Alcohol intake
– Smoking
– Sedentary lifestyle
– Being overweight
(Alanazi and Khan, 2018).

5. Figure 2: Structure of HER2 receptor
(Adapted from: Moasser, 2007).
HER2 Receptor
• HER2 is a receptor tyrosine kinase.
• Transmembrane glycoprotein having
a molecular weight of 185 kD.
• Encoded by the HER2 gene, located
in chromosome 17.
• Belongs to the four-membered HER
(ErbB/EGFR) family (HER1, HER2,
HER3, HER4).
• Does not have an identified ligand.
(Iqbal and Iqbal, 2014).

6. Figure 3:
Activation of
HER2 signalling
cascades
(Adapted from:
Lv et al., 2016).
Signalling Pathway

7. MAPK pathway
Figure 4: EGFR signaling pathway
in breast cancer (Adapted from:
Aziz and Aziz, 2013).
Figure 6: Adaptor proteins binding and Ras
activation (Adapted from: JJ Medicine, 2017).
Figure 5: RTK activation; A - Ligand binding and
receptor clustering, B - cross phosphorylation
(Adapted from: Slideplayer, no date).
A
B

8. MAPK pathway
• Growth factor binds to EGFR.
• Neighbouring receptors cluster.
• Cross phosphorylation of tyrosine residues of RTK.
• Binding of adaptor proteins – GRB2 and SOS.
• Activation of Ras  Raf  MEK  MAPK  transcription factors.
(Aziz and Aziz, 2013).
 EGFR - Epidermal Growth Factor Receptor
 RTK - Receptor Tyrosine Kinase
 GRB2 - Growth Factor Receptor Bound Protein 2
 SOS - Son of Sevenless
 RAF/MAPKKK - Mitogen-activated Protein kinase kinase kinase
 MEK/MAPKK - Mitogen-activated Protein kinase kinase
 ERK/MAPK - Extracellular Signal Regulated Kinase

9. Figure 7: PI3/AKT/mTOR pathway associated with breast cancer (Paplomata and Regan, 2014).
PI3/AKT/mTOR signaling

10. (Velloso et al., 2017).
PI3K / AKT / mTOR
• Ligand binding to the HER2 receptor associated heterodimers
(HER2/HER3) will activate the RTK by phosphorylation.
• Activated RTK can activate the PI3Ks (Phosphatidylinositol-3 kinases).
• These PI3Ks can phosphorylate PIP2 (Phosphatidylinositol 4,5-
bisphosphate) in the plasma membrane to PIP3 (Phosphatidylinositol-
3,4,5- triphosphate).
• De-phosphorylation of PIP3 back into PIP2 is regulated by tumor
suppressor known as PTEN.
• PIP3 recruits and activates protein kinase B (PKB) which is also known as
AKT by Phosphorylation.
• PKB or AKT can phosphorylate down stream proteins to regulate cell
growth, survival and apoptosis.

11. Mutations associated with
PI3/AKT/mTOR pathway
These mutations can induce HER2+ breast cancer:
1. AKT mutation
• In breast cancer, AKT mutations takes a prominent place where AKT1 can
promote cell proliferation by upregulation of S6 and cyclin D1, where as AKT2
stimulate migration of cancer cells and invasion via F actin and vimentin.
2. PI3KCA gene Mutation
• PI3KCA gene codes for P110α which is the catalytic subunit of PI3K [class IA].
P110α is important for duct formation duct branching and lactation eventually
for mammary gland development. So, all these functions become impaired.
(Paplomata and Regan, 2014).

12. 3. PTEN mutation or downregulation
• This can lead to the over stimulation of AKT without getting switched off,
which can furthermore enhance downstream protein function.
4. Upregulation of mTOR
• Due to upregulation of mTORC1 can increase cell metabolism and anabolic
cell growth by its action on protein S6K1 and 4EBP1.
(Paplomata and Regan, 2014).
Figure 8: Mutations associated
with PI3/AKT pathway that can
induce HER2+ breast cancer
(Velloso et al., 2017).

13. Pathogenesis
• HER2-positive breast cancer is characterized by the:
– Over-expression of the HER2 protein  corresponds with a more hostile disease.
– HER2 gene amplification
• Mechanism of HER2 activation in HER2+ tumors  Receptor overexpression.
• HER2 activation causes gene expression alterations mediated through changes
in transcription, translation and protein stability, which affect cell growth and
proliferation.
• Unlike its members of the epidermal growth factor family (EGF), the
extracellular domain of HER2 receptor has no identifiable activating ligand:
– Activation upon homo- or hetero-dimerization resulting in autophosphorylation of
tyrosine residues.
– Initiates a variety of signalling pathways;
• Mitogen-activated protein kinase (MAPK)
• Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)
(Oncology Nurses, 2018; Iqbal and Iqbal, 2014; Mitri et al., 2012).

14. HER2 receptor over-expression
h Cell surface HER2
receptors
h Receptor-receptor
interactions
Provokes sustained
tyrosine phosphorylation
Constant activation of the
signalling pathways
 MAPK
 PI3K
Enhances HER2 hetero-
dimerization with HER1 and HER3
h activation of the downstream
signalling pathways
h cell proliferation, survival,
differentiation, angiogenesis and
invasion
• Hetero-dimerization
generates more potent signals
than homo-dimers.
• Those with HER2 have high
ligand binding and signalling
potency.
Generally, after ligand-
mediated activation and
homo-dimerization
HER1 undergoes
endocytic degradation.
However,
HER1-HER2 hetero-
dimers evade endocytic
degradation
hHER1 membrane
expression and activity.
(Furrer et al., 2018; Iqbal and Iqbal, 2014; Ménard et al., 2000).

15. Differential diagnosis of
HER2 breast cancer
• Initial diagnostic test: palpation in the right breast. Confirmed the
presence of a lump.
• Recommended tests for further confirmation and accurate diagnosis:
1. Diagnostic Mammogram of right breast: An X-ray of the breast. Aims for
early detection of breast cancer. Able to detect the presence of non-
palpable tumours (<15mm) (Coleman, 2017).
2. Core needle biopsy: aims for early diagnosis of suspicious lesions. Tissue
from lesion is obtained using a needle. Sample obtained will be tested
histologically for HER2/neu expression and hormone receptor status.
3. PET Scan and CT scan: Positron Emission Tomography and Computerized
Tomography. Performed to observe the metastasis of the cancer or if the
treatments are effective.

16. 4. Analysis of serum tumour markers: Biomarkers found in serum that elevates in the
presence of a cancer. Provides details about the metastasis status of the cancer (Kabel, 2017).
• CEA (carcinoembryonic antigen): presence due to metastasis of breast cancer to colon,
lungs and liver.
• CA 15.3 and CA 125: presence of breast and ovarian cancer.
• Oestrogen receptor
• Progesterone receptor
• HER2/neu
5. Complete blood count: common and simplest test done during any diagnosis to detect
underlying conditions and health of the blood.
6. Biochemical tests
• Liver function test: provides information if the cancer has metastasized to the liver.
• Test for potassium, chloride and urea levels: to detect any abnormalities in the liver and
kidney.
• Test for calcium levels: to detect abnormalities in the kidneys or bones.

17.  IHC scores developed in 2013 by ASCO/CAP guidelines based on cell membrane staining
pattern is as below.
 Semi-quantitative and based on 4 classes (Krishnamurti et al, 2014).
• 0 and +1 - negative for HER 2 protein overexpression. No/weak membrane staining.
• +2 – equivocal for HER2 protein overexpression. Weak/incomplete circumferential
membrane staining in > 10% of the invasive tumour cells.
• +3 – positive for HER2 overexpression. Complete/uniform/intense circumferential
membrane staining in > 10% of the invasive tumour cells.
 IHC equivocal samples will then undergo FISH assay for further confirmation.
A B
Figure 9: IHC staining; A - displays normal levels
of HER2 receptor expression, B - displays
abnormal levels of HER2 receptor expression
(Iqbal and Iqbal, 2014).
IHC – Immuno Histochemistry

18.  More reliable, accurate and sensitive.
 Requires special equipment and special trained personnel. More tedious.
 Detects gene amplification.
 Scores of FISH assays according to ASCO/CAP 2013 are below.
Result HER2:CEP17 Gene copy
number
Positive > 2.2 > 6.0
Equivocal 1.8-2.2 4.0-6.0
Negative < 1.8 < 4.0
(Krishnamurti et al, 2014).
Figure 10: FISH Analysis; A - displays
normal copy numbers of HER2 gene,
B - displays abnormal copy numbers of
HER2 gene (Iqbal and Iqbal, 2014).
FISH - Fluorescence
In Situ Hybridization
A B

19. References
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