g-protein–coupled receptor gpr161 is overexpressed in breast cancer and is a promoter of cell...
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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 LaboratoryStony Brook University, NY
University of Toronto
Proceedings of the National Academies of Science, USAMarch 18, 2014
111:491-496
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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 prognosisand lack of targeted therapies
Tamoxifen family of drugs targets ERHerceptin targets HER2
To develop new therapies for TNBC,we need to understand its causes.
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Triple-Negative Breast Cancer (TNBC)
~15% of TNBCs are associated with BRCA1 or BRCA2 mutationsThese gene account for about most of familial breast cancers, ~5-10% of totalBoth 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)
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TGCA Screenshot
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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 GPCRagonists or antagonists
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GPCRs
Many inputs
Many targets
Nature Rev. Cancer 7:79
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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)
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GPR161 in TNBC
GPR161 is upregulated 2.2-fold in TNBC
Not upregulated in ER+ tumors (LumA/B)HER2+ tumors
Fig. 1A
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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
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GPR161 in TNBC
Fig. 1BC
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
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Fig. S1C
GPR161 in TNBC
For any type of TNBC, high GPR161 expression decreased time to relapse by 27%
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GPR161 in normal and malignant breast tissueBreast is a complicated tissue made up of many cell types. Which cell types express GPR161?
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GPR161 in normal and malignant breast tissue
Lactiferous Duct
Luminal Epithelial Cells
Myoepithelial Cells
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GPR161 in normal and malignant breast tissue
Fig. 1D
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
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GPR161 in normal and malignant breast tissue
Does this localization pattern change during cancer progression?
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GPR161 in normal and malignant breast tissueDoes this localization pattern change during cancer progression?
Fig. 1E
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GPR161 in normal and malignant breast tissue
What happens when GPR161 is overexpressed?
MCF-10A cells are immortalized breast epithelial cellsInfected with a retrovirus causing stable, mild GPR161 overexpressionControl 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
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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 lineLots of collagen, laminin, entactin and some growth factorsCultured for two weeks
Fig. 2B
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GPR161 in normal and malignant breast tissueWhat happens when GPR161 is overexpressed?
Fig. 2B
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GPR161 in normal and malignant breast tissueWhat happens when GPR161 is overexpressed?
Fig. 2C
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GPR161 in normal and malignant breast tissue
Similar effect with MDA-MB-361 cells
Fig. S1DE
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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
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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 assayEach cell line was normalized to its control
Fig. 2E
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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
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GPR161 in normal and malignant breast tissue
Is GPR161 required for proliferation of breast cancer cells?
shRNA to knockdown GPR161 expressionMTT assay
Fig. 2FGH
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GPR161 and mTOR
What pathway(s) is used by GPR161 to affect proliferation?
Reverse Phase Protein Array (RPPA) data from TGCAProteins from various cancers plated as an arrayIncubated with a specific antibodyQuantify differences across tumors
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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
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GPR161 and mTOR
Many of these proteins are in the mTOR pathway
Connects nutrient level and growth control
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GPR161 and mTOR
Fig. 3B
Examined phosphorylation state of key
proteins in MDA-MB-361 cells with or
without GPR161 overexpression
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GPR161 and mTOR
Fig. 3C
GPR161 leads to more S6 phosphorylation.
Is it mTOR-dependent? or could it be another kinase?
Rapamycin directly inhibits mTOR
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GPR161 and mTOR
Fig. 3D
Is mTOR important for the ability of GPR161 to induce proliferation?
MDA-MB-361 cells with or without GPR161 overexpressionwith or without rapamycin treatment
MTT assay
Conclusion?
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GPR161 and mTOR
Fig. 3EF
Is mTOR important for the ability of GPR161 to induce proliferation?
MCF-10A cells with or without GPR161 overexpression in Matrigelwith or without rapamycin treatment
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GPR161 and mTOR
Fig. 3
Is GPR161 upstream or downstream of mTOR?
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GRP161’s Effect on Cell Biology
Cells with elevated expression of GPR161 just look different in subconfluent cultures.
Controls formedcolonies withrounded edges.
GPR161 over-expressing cells showed sharp edges and projections.
Less adhesive?More invasive?
Fig. S2BC
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GRP161’s Effect on Cell Biology
Transwell migration assay
Insert 5,000 cells here
8mm filter
Count cells here after 24 h
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GRP161’s Effect on Cell Biology
Transwell migration assay
Two cell lines,with or without GPR161overexpression
Fig. 4A
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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
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GRP161’s Effect on Cell Biology
Invasive cells typically down-regulate Laminin-V
Laminin-V detected by IHC in redstructures are “disrupted”
Fig. 4C
Plasma Membrane
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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
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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-beadsremoves most plasma membrane fragmentsintracellular membranes remain
Suggests E-cadherin is significantly mislocalized
Fig. 4E
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GRP161’s Effect on Cell Biology
Fig. 4F
MDA-MB-361 cells
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GRP161’s Effect on Cell Biology
Fig. 4G
Human Tumors
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GRP161’s Effect on Cell Biology
Fig. S2E
AdditionalHumanTumors
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GPR161
?
Connecting GPR161 and mTOR
We concluded that mTOR is downstream of GPR161.
But how are they connected?
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Connecting GPR161 and mTOR
Hypothesis: GPR161 interacts with IQGAP1/b-Arrestin
IQGAP1 is found at E-Cadherin focal adhesions
IQGAP1 can interact with mTORonly if unphosphorylated
IQGAP1 is a oncogene for colorectal cancers
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Connecting GPR161 and mTOR
Does GPR161 alter IQGAP1 phosphorylation?
MCF-10A or MDA-MB-361 cells, with or without GPR161 overexpression
IP IQGAP1Western blot with an anti-phospho-serine antibody
Fig. 5A
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Connecting GPR161 and mTOR
Is GPR161 in a complex with IQGAP1 and b-Arrestin?
coIP from mouse 293T cellsexpressing FLAG-epitope tagged GPR161and myc-epitope tagged IQGAP1and sometimes HA-epitope tagged b-Arrestin-1 or b-Arrestin-2
Fig. 5BC
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Connecting GPR161 and mTOR
Fig. 5D
coIP from breast cancer cellsusing untagged proteins
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Connecting GPR161 and mTOR
Fig. 5EFG
Is IQGAP1 needed for GPR161 overexpression phenotypes?
Knockdown with shRNA in MDA-MB-361
MTT assay and transwell migration assay
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Connecting GPR161 and mTOR
Both GPR161 and IQGAP1 have been reported to be overexpressed in breast tumors.
Of 748 breast tumors in TGCA166 (22.2%) had amplified GPR16139 (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 bothMuch more than expected by chance
Fig. 5H