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G-protein–coupled receptor GPR161 is overexpressed in
breast cancer and is a promoter of cell proliferation and
invasion
Michael E. Feigin, Bin Xue, Molly C. Hammell, and Senthil K. Muthuswamy
Cold Spring Harbor Laboratory
Stony Brook University, NY
University of Toronto
Proceedings of the National Academies of Science, USA
March 18, 2014
111:491-496
Triple-Negative Breast Cancer (TNBC)
No expression of
• Estrogen Receptor (ER)
• Progesterone Receptor (PR)
• ErbB2 (EGF Receptor/HER2)
~25% of all breast cancers
Generally worse prognosis
and lack of targeted therapies
Tamoxifen family of drugs targets ER
Herceptin targets HER2
To develop new therapies for TNBC,
we need to understand its causes.
Triple-Negative Breast Cancer (TNBC)
~15% of TNBCs are associated with BRCA1 or BRCA2 mutations
These gene account for about most of familial breast cancers, ~5-10% of total
Both are involved with DNA repair
Origin of BRCA+ TNBCs is unclear
Analyzed patient tumor genomes in The Cancer Genome Atlas (TCGA)
Specifically looked for overexpressed G Protein Coupled Receptors (GPCRs)
TGCA Screenshot
GPCRs
Seven transmembrane domains
Receptors for many extracellular signals
Leads to the activation of a heterotrimeric G protein
Humans encode ~800 GPCRs (~4% of all genes!)
Very “druggable”
~30% of current drugs target a GPCR
agonists or antagonists
GPCRs
Many inputs
Many targets
Nature Rev. Cancer 7:79
GPR161 in TNBC
Seeking GPCRs that are overexpressed in TNBC
Can’t just look at genomic DNA sequence!
Seeking a change in expression not a mutation
Looked at RNA sequencing data (RNA-seq) at TGCA
cDNAs generated from tumor mRNA and sequenced
98 TNBC compared to 100 normal breast tissue samples (nonmatched)
Followed 366 GPCRs (all known to not be involved with the senses)
Seeking GPCRs that are over-represented (on enriched) in the RNA-seq data
45 GPCRs were upregulated significantly (at least 2-fold)
GPR161 in TNBC
GPR161 is upregulated 2.2-fold in TNBC
Not upregulated in
ER+ tumors (LumA/B)
HER2+ tumors
Fig. 1A
GPR161 in TNBC
GPR161 is upregulated 2.2-fold in TNBC
GPR161 is upregulated in other breast
cancer datasets:
Richardson Breast 2 Panel
(40 samples)
Farmer Breast Study
(49 samples)
Fig. 1A, S1AB
GPR161 in TNBC
For most of these samples, clinical data are available on the patient.
Did high levels of GPR161 expression correlate with relapse-free survival rates?
Compared highest and lowest quartile of GPR161 expression
Among basal type TNBC, high GPR161 expression decreased time to relapse by
113% for lymph node positive and 54% for all basal cancers
Fig. 1BC
GPR161 in TNBC
For any type of TNBC, high GPR161 expression decreased time to relapse by 27%
Fig. S1C
GPR161 in normal and malignant breast tissue
Breast is a complicated tissue made up of many cell types.
Which cell types express GPR161?
GPR161 in normal and malignant breast tissue
Lactiferous Duct
Luminal Epithelial Cells
Myoepithelial Cells
GPR161 in normal and malignant breast tissue
Normal human mammary gland tissue
DAPI binds DNA and fluoresces blue
E-cadherin detected by fluorescent IHC (marker for luminal epithelial cells)
GPR161 detected by fluorescent IHC (using a different fluor)
Three pictures of the
same field of view;
two images merged
Scale bar = 10mm
Fig. 1D
GPR161 in normal and malignant breast tissue
Does this localization pattern change during cancer progression?
GPR161 in normal and malignant breast tissue
Does this localization pattern change during cancer progression?
Fig. 1E
GPR161 in normal and malignant breast tissue
What happens when GPR161 is overexpressed?
MCF-10A cells are immortalized breast epithelial cells
Infected with a retrovirus causing stable, mild GPR161 overexpression
Control retrovirus is PIG (murine stem cell virus puromycin-IRES-GFP)
BT-474 are transformed cells from an IDC
MDA-MB-361 are cultured
from a breast tumor
that metastasized to the
brain.
Fig. 2A
GPR161 in normal and malignant breast tissue
What happens when GPR161 is overexpressed?
MCF-10A cells can be grown in 3D, leading to ducts.
Plastic plates are coated with Matrigel – extracellular matrix proteins secreted by a cell line
Lots of collagen, laminin, entactin and some growth factors
Cultured for two weeks
Fig. 2B
GPR161 in normal and malignant breast tissue
What happens when GPR161 is overexpressed?
Fig. 2B
GPR161 in normal and malignant breast tissue
What happens when GPR161 is overexpressed?
Fig. 2C
GPR161 in normal and malignant breast tissue
Similar effect with
MDA-MB-361 cells
Fig. S1DE
GPR161 in normal and malignant breast tissue
So how does GPR161 overexpression lead to multiacinar formation and filled lumens?
Does it cause hyperproliferation?
Cells cultured in 96-well plate followed by MTT assay
GPR161 in normal and malignant breast tissue
So how does GPR161 overexpression lead to multiacinar formation and filled lumens?
Does it cause hyperproliferation?
Cells cultured in 96-well plate followed by MTT assay
Each cell line was normalized to its control
Fig. 2E
GPR161 in normal and malignant breast tissue
So how does GPR161 overexpression lead to multiacinar formation and filled lumens?
Does it cause hyperproliferation?
Ki67 staining as a marker of proliferation
In controls, Ki67+ cells in
6.3% of acini.
In GPR161 overexpressors,
Ki67+ cells in 57.5% of acini.
Fig. 2D
GPR161 in normal and malignant breast tissue
Is GPR161 required for proliferation of breast cancer cells?
shRNA to knockdown GPR161 expression
MTT assay
Fig. 2FGH
GPR161 and mTOR
What pathway(s) is used by GPR161 to affect proliferation?
Reverse Phase Protein Array (RPPA) data from TGCA
Proteins from various cancers plated as an array
Incubated with a specific antibody
Quantify differences across tumors
GPR161 and mTOR
What pathway(s) is used by GPR161 to affect proliferation?
Reverse Phase Protein Array (RPPA) data from TGCA
Many alterations, including phospho-EIF4BP1 and phospho-RPS6KA1 and others
Fig. 3A
GPR161 and mTOR
Many of these proteins are in the mTOR pathway
Connects nutrient
level and
growth control
GPR161 and mTOR
Examined phosphorylation state of key
proteins in MDA-MB-361 cells with or
without GPR161 overexpression
Fig. 3B
GPR161 and mTOR
GPR161 leads to more S6 phosphorylation.
Is it mTOR-dependent? or could it be another kinase?
Rapamycin directly inhibits mTOR
Fig. 3C
GPR161 and mTOR
Is mTOR important for the ability of GPR161 to induce proliferation?
MDA-MB-361 cells with or without GPR161 overexpression
with or without rapamycin treatment
MTT assay
Conclusion?
Fig. 3D
GPR161 and mTOR
Is mTOR important for the ability of GPR161 to induce proliferation?
MCF-10A cells with or without GPR161 overexpression in Matrigel
with or without rapamycin treatment
Fig. 3EF
GPR161 and mTOR
Is GPR161 upstream or
downstream of mTOR?
Fig. 3
GRP161’s Effect on Cell Biology
Cells with elevated expression of GPR161 just look different in subconfluent cultures.
Controls formed
colonies with
rounded edges.
GPR161 overexpressing cells
showed sharp
edges and
projections.
Less adhesive?
More invasive?
Fig. S2BC
GRP161’s Effect on Cell Biology
Transwell migration assay
Insert 5,000 cells here
8mm filter
Count cells here after 24 h
GRP161’s Effect on Cell Biology
Transwell migration assay
Two cell lines,
with or without GPR161
overexpression
Fig. 4A
GRP161’s Effect on Cell Biology
MCF-10A cells, with or without GPR161 overexpression
Grown in 1:1 Matrigel:Collagen for two weeks
Fig. 4B
GRP161’s Effect on Cell Biology
Invasive cells typically down-regulate Laminin-V
Laminin-V detected by IHC in red
structures are “disrupted”
Plasma Membrane
Fig. 4C
GRP161’s Effect on Cell Biology
Cell-cell adhesion is mediated by E-Cadherin (among many other proteins)
Measure E-Cadherin levels in MCF-10A cells
“modestly reduced”
Fig. 4E
GRP161’s Effect on Cell Biology
Concanavalin A (Con A) is a plant protein that binds certain carbohydrate groups
that are abundant on glycoproteins and glycolipids
Total Cell Lysase (TCL) was run over ConA-beads
removes most plasma membrane fragments
intracellular membranes remain
Suggests E-cadherin is significantly mislocalized
Fig. 4E
GRP161’s Effect on Cell Biology
MDA-MB-361 cells
Fig. 4F
GRP161’s Effect on Cell Biology
Human Tumors
Fig. 4G
GRP161’s Effect on Cell Biology
Additional
Human
Tumors
Fig. S2E
Connecting GPR161 and mTOR
GPR161
We concluded that mTOR is
downstream of GPR161.
But how are they connected?
?
Connecting GPR161 and mTOR
Hypothesis:
GPR161 interacts with IQGAP1/b-Arrestin
IQGAP1 is found at E-Cadherin focal adhesions
IQGAP1 can interact with mTOR
only if unphosphorylated
IQGAP1 is a oncogene for colorectal cancers
Connecting GPR161 and mTOR
Does GPR161 alter IQGAP1 phosphorylation?
MCF-10A or MDA-MB-361 cells, with or without GPR161 overexpression
IP IQGAP1
Western blot with an anti-phospho-serine antibody
Fig. 5A
Connecting GPR161 and mTOR
Is GPR161 in a complex with IQGAP1 and b-Arrestin?
coIP from mouse 293T cells
expressing FLAG-epitope tagged GPR161
and myc-epitope tagged IQGAP1
and sometimes HA-epitope tagged b-Arrestin-1 or b-Arrestin-2
Fig. 5BC
Connecting GPR161 and mTOR
coIP from breast cancer cells
using untagged proteins
Fig. 5D
Connecting GPR161 and mTOR
Is IQGAP1 needed for GPR161 overexpression phenotypes?
Knockdown with shRNA in MDA-MB-361
MTT assay and transwell migration assay
Fig. 5EFG
Connecting GPR161 and mTOR
Both GPR161 and IQGAP1 have been reported to be overexpressed in breast tumors.
Of 748 breast tumors in TGCA
166 (22.2%) had amplified GPR161
39 (5.2%) had amplified IQGAP2
Are the same tumors overexpressing both genes?
Or do breast tumors overexpress one gene and not the other?
13 show overexpression of both
Much more than expected by chance
Fig. 5H