HER2 Splice Variants and Their Relevance in CREB1 Mediated Response.

Better understanding the pharmacogenomic role of different HER2 variants on CREB1 in Breast Cancer Cell Lines.

Kathleen Harrison, TA; Stephanie PreziosoCSB349H1University of Toronto

ObjectivesBackgroundpharmacogenomicsHer2 in cancerDrug treatments

Research ProposalMethodologyExpected resultsFuture work

ReferencesSpecial thanks

● Heterodimer membrane glycoprotein

● Cell cycle control, proliferation and survival

● Oncogenic signaling ○ RAS, RAF, MAPkinase, PI3K, AKT, mTOR,

and STAT, EGFR, HER-2, Raf, AKT, and ERK. -- FAK, Src kinase

● Effect genes such as ○ MMP1, ANGPTL4, MET, CD44, PLAUR,

EPHA2, ITGA2, ITGFB, TGFA, and IL-11

What is HER2?

HER2+ in Breast Cancer● More at risk of

○ Poor prognosis○ Increased tumor growth○ Proliferation ○ Metastasis potential○ Resistance

● Overexpressed in 20-30% of patients

● Anti-HER2 treatments only effective ~50%

HER2 Receptor Variants● ∆16HER2 no exon16 of the ERBB2

gene

● Homodimers --> self autophosphorylation

● Increased metastatic potential

● p95HER2 --> 611-CTF & 687-CTF.

● CTFs lack an ECD - metastatic

● Occurs in ~25% of HER2 tumors

Pharmacogenomics and Oncology

● Disease-specific neo-epitopes

● Biomarkers to improve outcomes

CREB Diagnostic marker?

● Genetic variation could explain 20-95% of outcome differences

Lapatinib and HER2

-Tyrosine Kinase Inhibitor of HER2

-Prevents autophosphorylation and proliferation

-Lapatinib is concentration dependent and can cause extensive side effects

-Trastuzumab more effective with Lapatinib

Trastuzumab and HER2

-First drug Treatment to have a clinical application in breast cancer

-Recombinant Monoclonal-Antibody binds to domain four of the extracellular domain of HER2

-Trastuzumab triggers an immune response (Antibody Dependent Cell-Mediated Cytotoxicity)

-Δ16HER2 splice variants are resistant to Trastuzumab

-The Loss of PTEN renders HER2+ cells resistant to trastuzumab

Pertuzumab and HER2

Monoclonal Antibody Binding to Junctions of domains 1-3 and triggers an immune response

Inhibits HER2 interactions with other receptors

Block HRG signalling

Ability to block Dimerization is independent of HER2 levels

Pertuzumab shows low side effects ( e.g. diarrhea, vomiting and skin rash)

Pertuzumab Vs . Trastuzumab-Pertuzumab effective in trastuzumab-resistant cells

Pertuzumab and Trastuzumab share the same sequence except in the CDR regions

-Drugs work more efficiently together than separately

MethodologyOutline: Experiment Part 1

Utilizing 2 HER2+ Human Breast Cancer Cell (BCC) Lines from:

● AU565 cell line, (Highly overexpressing HER2+) ● BT-474 cell line, (Luminal B, Poor differentiation / High

Grade)○ For each BCC line, we will create 3 variants

■ ∆16HER2+ ■ CTF-687+ ■ Nonsense code (transduction control).

Methodology - Modifying Cell Lines● cDNA from HER2 Splice variants isolated and cloned into

construct.

● Transfection and transformation, via a viral vector, of the AU565 and BT-474 BCC lines

● Prior to transfection, eachline will be separately seeded into eight (8) dishes and incubated to reacha high level of confluency

Methodology - Modifying Cell Lines● Three (3) constructs will be used, containing

∆16HER2 cDNA, CTF-687 cDNA or non-sense code (NC)

● Each construct will also include exogenous promoter sites for the splice variant and a GFP reporter along with an endogenous promoter for a sequence encoding for antibiotic resistance.

● Resulting variants: ■ AU565-∆16+■ AU565-C687+■ AU565-NC■ BT-474-∆16+■ BT-474-C687+ ■ BT-474-NC

MethodologyOutline: Experiment Part 1 Continued

Test each variant line, with and without TF specific for promoter, using the following procedures to establish baseline:

○ Cell Proliferation assay and migration/invasion assay(adapted from Steven et al., 2013)

○ CREB1 mRNA levels, via Real-Time RT-PCR (qRT-PCR)○ pCREB1 levels, via Western blot

Unmodified AU565 and BT-474 group tested as control

Methodology

Outline: Experiment Part 2

For each BCC line, we will test the two transfected variants and an unmodified control, all with the TF specific for our promoter along with different drug combinations of Lapatinib, Trastuzumab and Pertuzumab as outlined:

Each variant will be exposed to six (6) drug combinations

Lapatinib (L) Pertuzumab (P) Trastuzumab (Tr)

L L P/L Tr/L

P P Tr/P

Tr Tr

Anticipated Results

● Efficacy of drugs evaluated by ability to decrease levels of:○ cell proliferation

■ XTT assay (Colorimetry)○ invasion/migration

■ Scratch assay○ CREB RNA

■ qRT-PCR (Real time Reverse Transcriptase - Polymerase Chain Reaction)

○ CREB protein■ Western Blot with

Fluorescence labeled probe

Figure from Mitas et al, 2001

Figure from Stephen et al, 2013

AU565-∆16+ & BT-474-∆16+

● Trastuzumab - little to no effect because binding domain is not present○ also lack cytotoxic T cells

● Low levels of cell proliferation, invasion/migration, CREB RNA and CREB protein when treated with Lapatinib or Pertuzumab (good)

● Lowest levels of cell proliferation, invasion/migration, CREB RNA and CREB protein when treated with Lapatinib and Pertuzumab (best)

AU565-C687+ & BT-474-C687+

● Absence of wild type extracellular domain○ Trastuzumab and Pertuzumab expected to have

the least effect on levels of cell proliferation, invasion/migration, CREB RNA and CREB protein

○ Lapatinib will induce decreased levels of cell proliferation, invasion/migration, CREB RNA and CREB protein

Unmodified Variant AU565 and BT-474○ We expect Pertuzumab and Lapatinib together

to work best for the unmodified variant■ Induce lowest levels in the cell proliferation

assay, migration assay as well as lowest CREB RNA and CREB protein

○ Trastuzumab acts by binding to HER2 receptor and recruiting cytotoxic T cells which are absent in vitro

Results summarized● ∆16HER2+ variant: Pertuzumab and Laptinib together will induce

lowest levels of CP/IM/RNA/Protein

● CTF-687+ variant: Lapatinib alone will induce lowest levels of CP/IM/RNA/Protein

● Unmodified: Pertuzumab and Lapatinib together will induce lowest levels

Further Research/ApplicationsPerform in vivo to establish toxicity of drug combinations

● Should be enhanced in vivo due to interaction with immune system, but other cell types may be affected.

Test more breast cancer cell lines● Cancers involve many receptors (i.e. ER, PgR) affecting drug response. ● Establish HER2 targeting treatments vs traditional treatment strategies

Used to enhance personalization of HER2+ cancer treatment.● Breast cancer treatment could include HER2 variants to aid physicians.

Apply to other cancers that involve HER2 ● Uterine, ovarian and stomach cancers are all exacerbated by HER2

overexpression.

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