Triple-Negative Breast Cancer (TNBC)

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

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.

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)

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 GPCRagonists 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

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

Fig. S1C

GPR161 in TNBC

For any type of TNBC, high GPR161 expression decreased time to relapse by 27%

GPR161 in normal and malignant breast tissueBreast 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


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


Does this localization pattern change during cancer progression?

GPR161 in normal and malignant breast tissueDoes this localization pattern change during cancer progression?

Fig. 1E


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


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

GPR161 in normal and malignant breast tissueWhat happens when GPR161 is overexpressed?

Fig. 2B

GPR161 in normal and malignant breast tissueWhat happens when GPR161 is overexpressed?

Fig. 2C


Similar effect with MDA-MB-361 cells

Fig. S1DE


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



Does it cause hyperproliferation?

Cells cultured in 96-well plate followed by MTT assayEach cell line was normalized to its control

Fig. 2E



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


Is GPR161 required for proliferation of breast cancer cells?

shRNA to knockdown GPR161 expressionMTT assay

Fig. 2FGH

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

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

Fig. 3B

Examined phosphorylation state of key

proteins in MDA-MB-361 cells with or

without GPR161 overexpression

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

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?

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

GPR161 and mTOR

Fig. 3

Is GPR161 upstream or downstream of mTOR?

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


Transwell migration assay

Insert 5,000 cells here

8mm filter

Count cells here after 24 h


Transwell migration assay

Two cell lines,with or without GPR161overexpression

Fig. 4A


MCF-10A cells, with or without GPR161 overexpression

Grown in 1:1 Matrigel:Collagen for two weeks

Fig. 4B


Invasive cells typically down-regulate Laminin-V

Laminin-V detected by IHC in redstructures are “disrupted”

Fig. 4C

Plasma Membrane


Cell-cell adhesion is mediated by E-Cadherin (among many other proteins)

Measure E-Cadherin levels in MCF-10A cells

“modestly reduced”

Fig. 4E


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


Fig. 4F

MDA-MB-361 cells


Fig. 4G

Human Tumors


Fig. S2E

AdditionalHumanTumors

GPR161

?

Connecting GPR161 and mTOR

We concluded that mTOR is downstream of GPR161.

But how are they connected?


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


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


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


Fig. 5D

coIP from breast cancer cellsusing untagged proteins


Fig. 5EFG

Is IQGAP1 needed for GPR161 overexpression phenotypes?

Knockdown with shRNA in MDA-MB-361

MTT assay and transwell migration assay


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

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Triple-Negative Breast Cancer (TNBC)