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DEAR1 is a Chromosome 1p35 Tumor Suppressor and Master Regulator of TGFβ-Driven Epithelial-Mesenchymal Transition

Nanyue Chen1, Seetharaman Balasenthil1, Jacquelyn Reuther1*, Aileen Frayna1,

Ying Wang1, Dawn S. Chandler1, Lynne V. Abruzzo2, Asif Rashid3, Jaime Rodriguez4,

Guillermina Lozano1, Yu Cao1, Erica Lokken1, Jinyun Chen5, Marsha L. Frazier5,

Aysegul A. Sahin3, Ignacio I. Wistuba4, Subrata Sen4*,

Steven T. Lott1, and Ann McNeill Killary1*

Departments of 1Genetics, 2Hemotopathology, 3Pathology, 4Translational Moleclar Pathology, and 5 Epidemiology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030,

USA. * The Human and Molecular Genetics Program, The University of Texas, Graduate School of

Biomedical Science at Houston, Houston, Texas.

(Supplementary Information)

Supplementary Methods

Cell Culture and Reagents: 76N-E6, MCF10A, MCF7 and U2OS were authenticated by STR

profiling at the characterized cell line core facility at MD Anderson Cancer Center. MDA-MB-

231, HEK293T, HeLa, HPDE, HPDE/KRas, PANC48, MIAPaCa2 and PANC1 have not been

authenticated.

Human Specimens: All human tissues were obtained without identifiers from the MD Anderson

Cancer Center tumor bank according to the IRB protocol.

Plasmids and antibodies: DEAR1 vectors and antibody were previously described (1). The

DEAR1 mutants and deletion constructs were generated by PCR and inserted in the pCMV-HA

vector. Anti-cleaved caspase 3, anti-phospho-SMAD3, anti-SMAD3 (for Western), anti-SMAD2

and anti-Snail2 (Slug) antibodies were obtained from Cell Signaling. Phospho-SMAD3 antibody

for immunostaining was from Millipore and anti-SMAD3 for immunohistochemistry was from

Invitrogen. All SMAD3 and phospho-SMAD3 antibodies were confirmed to have no

crossreactivity to SMAD2 by western in our laboratory. Antibodies for Vimentin, HA-tag, Myc-

tag, Flag-tag and β-actin were from Sigma. Antibodies for E-Cadherin, β1-Integrin and N-

Cadherin were from BD Biosciences.

Generation of DEAR1 knockout mouse. The targeting vector was constructed to contain a

5’fragment of DEAR1 intron 1, the PCKneoNTRtkpA cassette and a 3’fragment containing

DEAR1 intron 3 through intron 4 (Supplementary Fig. S1A). Following electroporation of the

targeting vector into ES cells from a 129Sv/Ev agouti male, ES cells were grown in selective

media and southern analyses performed to identify correctly targeted clones in which exon 1 of

Dear1 was replaced by a PCKneo cassette. One of 96 ES clones contained the correctly

incorporated targeting vector and was then injected into wild type C57BL6 blastocysts to

produce four chimeric mice, three of which successfully transmitted the recombinant allele

through the germline when crossed with C57BL6 wild types (Fig. 1A). Seven of 12 of the

agoutis generated were F1 heterozygotes. The resulting heterozygous mice, four females and

three males, were crossed to produce over 200 wild type, heterozygous and knockout progeny.

Generation of stable DEAR1-shRNA cell lines: Generation of stable DEAR1-shRNA clones in

76N-E6 HMECs was previously described (1). Following same protocol, stable DEAR1-shRNA

clones were generated in MCF10A. MISSION® shRNA lentiviral vectors expressing nontarget

control shRNA or DEAR1 shRNAs and packaging vectors were purchased from Sigma

(NM_018207). The sequences of DEAR1-siRNA were: 936-CGACTGCACCATTGTGGCT-

TACGGCAACTT and 1151-CGGCTTCTACTGCATCGTGAT. Cotransfection of retroviral and

packaging vectors into HEK293T packaging cells for production and packaging of retroviruses

was performed according to the manufacturer's recommendations. The supernatant containing

virus was harvested and filtered 48h-72h after transfection. Viral supernatant was infected into

MCF10A cells in the presence of 8 µg/ml hexadimethrine bromide. Stable clones were selected

using puromycin (2µg/ml).

Generation of SMAD3-shRNA cell lines: pRetroSuper-GFP SMAD3 shRNA plasmids (2)

(Addgene plasmid 15723) were transfected in 293T cells and the retrovirus was collected and

used to infect DEAR1-KD 76NE6 and DEAR1-KD MCF10A cells. Cells infected were sorted

twice with flow cytometry to enrich GFP-expressing cells (>99%). SMAD3 protein levels were

verified by Western (Fig. 6C).

Database Analysis: (See Supplementary Methods). Databases including cBIO (MSKCC) (3),

International Cancer Genome Consortium (ICGC), CONAN (Wellcome-Sanger), Catalogue of

Somatic Mutations in Cancer (COSMIC) (Wellcome-Sanger), and Oncomine were screened for

information detailing genomic alterations and mRNA/protein expression in TCGA cohorts, cell

lines, and other published papers. 1000 Genomes Project data, accessed by SNAP (Broad

Institute), along with dbSNP database were checked to verify novelty of mutations. ConSeq

(V1.1) was used to determine amino acid residue conservation. Mutation functionality

assessment was done via PolyPhen2 software (V2.2.2). Survival curves were generated by cBio,

using Kaplan-Meier analysis through querying complete tumor sets in the BRCA cohort for

DEAR1 heterozgyous loss, as well as SNAI1, SNAI2, TWIST1 and TWIST2 genetic alteration.

Alteration of SNAI1/2 and TWIST1/2 includes amplification, upregulation of mRNA/protein

expression (if applicable) greater than two standard deviations from the mean.

RNA Extraction and Q-RT-PCR assay: Total RNA from cultured cells and mouse tissues was

extracted using a TRIzol kit (Invitrogen) followed by use of a Absolute Mini-RNA kit

(Stratagene). Equal amounts of total RNA from each sample were reverse transcribed to cDNA

using the RT kit (Applied Biosystems). Primers and probes for all tested genes were from

Taqman® assays (Applied Biosystems) and quantitative real-time PCR was performed with a

PRISM 7500 (Applied Biosystems). The relative mRNA levels were normalized to 18S rRNA

using the comparative Ct method.

Transient transfection and reporter gene assays: HEK293T cells were seeded 20–24 h prior to

transfection in 24-well plates at 3 x 104 cells/well as described previously (4). Plasmid DNAs (in

quantities noted for each experiment) with FuGene(HD) transfection reagent (1ug:3ul) (Roche

Applied Science) were added to each well. At 24 h after transfection, the cells lysed in lysis

buffer (Promega) and luciferase activity was measured using a Monolight 2010 luminometer

(Turner BioSystems).

IP and Western: For immunoprecipitation following ectopic expression, Myc-tagged SMADs

and HA/DEAR1 were transiently co-transfected into HEK293T cells. After 24 hours, cells were

lysed in RIPA buffer or m-Per buffer (Pierce). After centrifuging, the supernatants were

incubated with rabbit-anti-HA (Sigma) over night and proteins were pulled down by protein A/G

agarose (SantaCruz Biotechnology). Beads were washed 3 times with PBS (containing 1%

TRITON-100 and 0.1% SDS) and then dissolved in 30ul of 2x SDS sample buffer for Western

blotting. For immunoprecipitation of endogenous protein, cells grown to 80% confluency were

lysed in m-Per buffer, and rabbit anti-SMAD3 (Cell Signaling) was used to pull down. For

Western blotting, equal amounts of protein per lane were loaded on 4-20% SDS–PAGE gradual

gels (Invitrogen), transferred to PVDF membranes, and analysed using different antibodies.

Immunohistochemistry: Immunohistochemistry was performed as previously described (1).

Inventory of Supplementary Figures

Figure S1: Related to Figure 1









Figure S10-12: Related to Figure 5


Video 1: Related to Figure 2




Legends for Supplemental Figures and Movies

Figure S1. Construct and identification of DEAR1 knockout mice. (A) Schematic

representation of DEAR1 knockout strategy. (B), Quantitative RT-PCR analysis of DEAR1

expression in lung tissues from Dear1+/+ and Dear1-/- mice. RNA was extracted and purified

with minRNA kit (Qiagen). Q-PCR was performed using TaqMan (Applied Biosystems). (C)

survival curve and (D) average survival time of DEAR1 wild type (n=52), heterozygous (n=92)

and knockout (n=62) mice. (E) Immunohistochemistry stain for DEAR1 in sections of Dear1+/-

tumors. (left) Lung tumor showing DEAR1 positive staining from Dear1+/- mice (40x); (right)

Breast adenocarcinoma showing DEAR1 negative staining in Dear1+/- mice (40x). (F) PCR

showing genotyping of tumor as well as normal tissues of Dear1+/- mice. DNA was extracted

from samples in formaldehyde fixed paraffin-embedded sections. M: DNA ladder; N: normal

tissue; T: tumor tissue. The positive control (+) is from a Dear1 wild type mouse tail.

Figure S2. DEAR1 LOH in various cancer cell lines as shown by CONAN-copy number

analysis (Sanger Institute) software. (A) LOH within chromosome 1p31 to 1p36 involving

DEAR1 in CONAN cell lines with empty boxes corresponding to loss of alleles and colored

boxes indicative of retention of alleles. The x axis indicates the genomic interval within the p

arm that is deleted and the y axis indicates the tissue type. Each line represents an individual cell

line. (B) Table summarizing data visualized in (A) and describing the percentage of LOH of

DEAR1 in multiple cancer types in the CONAN database.

Figure S3. DEAR1 Expression in human pancreatic cancer tissues. (A)

Immunohistochemical analysis of DEAR1 expression in a pancreatic tissue microarray. Strong

DEAR1 expression was observed in normal pancreas (top, left) and with varied staining

intensities in pancreatic adenocarcinoma (remaining panels). (B) Western analysis of DEAR1

expression in normal and pancreatic cancer cell lines.

Figure S4. Quantitative RT-PCR analysis of DEAR1-shRNA knockdown clones. All clones

were collected in TRIzol while growing exponentially. RNA were extracted following TRIzol

instruction and purified with minRNA kit (Stratagene). Q-PCR was performed using TaqMan

(Applied Biosystems).

Figure S5. 3D culture of DEAR1-KD clones. Cells were plated in 8-well cell culture chambers

at 5000 cells /well on the top of matrigel in the presence or absence of TGFβ (2ng/ml). Phase

contrast images of parent 76N-E6, DEAR1-shRNA clones (DshRs) and control-shRNA clone

(CshRs) with or without TGFβ (2 ng/ml) in 3D culture at indicated times.

Figure S6. (A) Morphologic alterations in DEAR1-shRNA immortalized HMEC clones (76N-

E6 cells) (DshR) as compared to control wild type DEAR1 clones (CshR). Cells were treated

with or without TGFβ (4 ng/ml) for 5 days. (B) Trace of cell migration in 3D culture from 72

hours time-lapse experiment. (C) Migration of DEAR1-shRNA clone (DshR) and control clone

(CshR) cultured in matrigel with or without TGFβ by time-lapse live image. The average

migration distance per cell (30-40 cells per field) in the entire 72 hour period was calculated. The

values showed are mean ± 1 SD of 5 fields. (D) Quantification of wound healing assay results

from DEAR1-KD (10A/DshR) and control clones (10A/CshR) in the MCF10A cell background

with or without TGFβ treatment (2ng/ml) for 24 hours. (E) Wound healing assay of DEAR1-

KD2 (shRNA targeting a different region of DEAR1 (DshR-KD2)) and control clones (CshR) in

the 76N-E6 cell background with or without TGFβ treatment (2ng/ml) for 24 hours. (F) Wound

assay in U2OS cells. Cells were transiently transfected with DEAR1 or vector control. After

incubation overnight, cells were scratched and treated with or without 2ng/ml TGFβ.

Figure S7. (A) Western analysis of EMT markers in DEAR1-shRNA knockdown MCF10A

clone (10A/DshR) and control clone (10A/CshR) with or without 4 ng/ml TGFβ treatment for 4

days. (B) The effect of DEAR1-shRNA on vimentin expression. DEAR1-shRNA clones (DshR)

and control clones (CshR) of 76N-E6 HMECs were treated with or without TGFβ (4 ng/ml) for 4

days. The cells were stained by immunofluorescence with anti-Vimentin antibody. (C) Western

analysis of EMT markers in DEAR1-shRNA KD2 clone (DshR-KD2, targeting a different region

of DEAR1) or control clone (CshR) of 76N-E6 cells with or without 4 ng/ml TGFβ treatment for

4 days. (D) Quantitative RT-PCR analysis of EMT markers in DEAR1-shRNA knockdown

clones. Cells were treated with or without TGFβ (4ng/ml) for 3h and 40h, then, were collected in

TRIzol. RNA was then extracted according to manufacturer’s instructions and purified using a

minRNA kit (Stratagene). Q-PCR was performed using TaqMan (Applied Biosystems).

Figure S8. (A) DEAR1 negatively regulates TGFβ signal transduction in MDA-MB-231 breast

cancer cell line and HeLa cervical cancer cell line. DEAR1 and CAGA12 plasmids were co-

transfected in cultured cells. After 24 hours, cells were treated with or without TGFβ (1 ng/ml)

for 24 hours and luciferase activity was measured. (B) DEAR1 negatively regulates

TGFβ/SMAD3 signal transduction. Cells were co-transfected with CAGA12 or PAI-1 reporter,

Myc/SMAD3 and HA/DEAR1 plasmids. After 24 hours, cells were treated with or without

TGFβ (1 ng/ml) for 24 hours and luciferase activitly was measured. (C) Dose dependent

inhibition of the TGFβ/SMAD3 pathway by DEAR1. Increasing concentrations of HA/DEAR1

plasmids were co-transfected with Myc/SMAD3 plasmids and CAGA12 luciferase reporter in

HEK293T cells. After 24 hours, cells were treated with or without TGFβ (1 ng/ml) for 24 hours

and luciferase activity was measured.

Figure S9. (A) Western analysis of SMAD3 in DEAR1-shRNA KD2 clone (DshR-KD2,

targeting a different region of DEAR1) and control clone (CshR) of 76N-E6 cells with or without

4 ng/ml TGFβ treatment for 4 days. (B) co-immunoprecipitation of DEAR1 and SMAD3

domains. Flag-SMAD3/MH1, Flag-SMAD3/L&MH2 and empty vector were cotransfected into

293T cells with HA/DEAR1 plasmids. DEAR1 was pulled down with anti-HA antibody and

anti-Flag was probed to detect SMAD3. (C) High magnification deconvolution confocal images

of immunofluorescence staining of HA/DEAR1 (in green) and Flag/SMAD3/MH1 (SMAD3/N)

and Flag/SMAD3/L&MH2 (SMAD3/L&C) (in red). HA/DEAR1 was cotransfected with

Flag/SMAD3 domains into U2OS cells. Yellow arrows showed colocalization of DEAR1 and

SMAD3. (D) DEAR1 expression inversely correlates with SMAD3 expression in early onset

breast cancer tumor/normal samples. Western analysis (left panel) and density quantification

(right panel) of DEAR1 and SMAD3 from paired breast cancer tissue samples. Tissues were

lysed in 1% SDS sample buffer and purified with methanol and chloroform and then dissolved in

1x SDS sample buffer. Protein amount was measured using the Bradford assay (Pierce). Equal

protein amount was loaded in each well for Western and confirmed by Coommasie Blue stain of

gels.

Figure S10. DEAR1 and SMAD3 IHC staining in human breast cancer tissues. a and d,

normal ducts with invasive carcinoma, arrow head indicates normal ducts; b and e, normal ducts

and invasive carcinoma; c and f, invasive carcinoma. Original magnification: a and d, 40x; b, c, e

and f; 200x. Strong staining for DEAR1 was observed in normal ducts (b, arrowhead) with

tumor heterogeneity observed in invasive cancer (moderate staining, empty arrowhead) (b) and

in (c) invasive carcinoma demonstrating negative staining for DEAR1. In the same section

SMAD3 staining appears slightly upregulated in invasive adenocarcinoma (empty arrowhead)

surrounding normal ducts (e, arrowhead) with strong staining (f) in a different region for

SMAD3 corresponding with the same region showing loss of expression of DEAR1 (c).

Figure S11. DEAR1 and SMAD3 IHC staining in human breast cancer tissues. DEAR1

and SMAD3 IHC staining in the normal ducts (a,d), DCIS (b,e) and invasive carcinoma (c,f)

from the same individual and located in the same histologic section. Original magnification,

200x. Strong DEAR1 staining was observed in the normal ducts (a) compared to heterogeneous

expression with some cells showing strong staining but overall much lower expression compared

to normal ducts in the DCIS and invasive carcinoma (b and c). Strong SMAD3 staining was

observed in the DCIS (e) and invasive carcinoma (f) compared to normal ducts (d).

Figure S12. (A) DEAR1-KD increased the nuclear accumulation of phospho-SMAD3 in

immortal HMECs. Low magnification deconvolution confocal images of immunofluorescence

staining of phospho-SMAD3 in DEAR1-shRNA 76N-E6 cells. DEAR1-shRNA clone (DshR)

and control clone (CshR) were plated on glass coverslips in 24-well plates. After 16 hours in

culture, cells were serum-starved with 1:10 diluted D-Medium for 24 hrs. After treatment with or

without 2 ng/ml TGFβ for 1 hour, cells growing on coverslips were fixed and immunostained

with anti-phosphor-SMAD3 (Millipore). All images were photographed using the same exposure

conditions for comparison of images. (B,C) HA/DEAR1 colocalizes with endogenous SMAD3

in MCF7 cells. (B). Stacked confocal images (from Fig. 5D) of one cell expressing HA/DEAR1

which was three-dimensionally reconstructed using the colocalization module of the Imaris

imaging system which measures the entire confocal stack for the intensity of each label per voxel

(or 3D pixel) unit. HA/DEAR1 is shown in green and SMAD3 is shown in red with grid units

equal to 4 um; (C) Iso-surface rendition obtained from the stacking images using Imaris. The

green patches represent HA/DEAR1 based on its density voxel. Then, the number of the voxels

of red color (SMAD3 signal) was calculated inside each patch. All patches corresponding to

HA/DEAR1 which contained greater than 1000 voxels of SMAD3 were shown in yellow,

representing colocalization of the two proteins.

Figure S13. (A) Western analysis of SMAD3 inhibitor, SIS3, on TGFβ-induced EMT

markers in MCF10A DEAR1-KD and control clones. DEAR1-shRNA MCF10A clone

(DshR) and control clone (CshR) were plated in 6-well plates with indicated SIS3 concentrations.

After 4-hour incubation, cells were treated with or without TGFβ (4ng/ml) for 3 days. Cells

were dissolved in 1x SDS sample buffer and protein amount was measured using the Bradford

assay (Pierce). Equal protein amount was loaded in each well for Western. (B) Wound assay in

DEAR1-shRNA 76N-E6 cells treated with TGFβ and SMAD3 inhibitor (SIS3). DEAR1-

shRNA clones (DshR1 and DshR2) and control clones (CshR1 and CshR2) were plated in 6-well

plates. Cells were scratched at 90% confluence and then, treated with 2ng/ml TGFβ or TGFβ

plus SMAD3 inhibitor, SIS3, for 24 hours.

Videos: Time lapse recording of 3D culture of 76N-E6 HMECs depicts extensive cell

migration though 3D cultures in DEAR1 knockdown HMECs in the presence of TGFβ.

Video 1. Control shRNA clone without TGFβ treatment. Video 2. Control shRNA clone with 2

ng/ml TGFβ treatment. Video 3. DEAR1-shRNA clone without TGFβ treatment. Video 4.

DEAR1-shRNA clone with 2 ng/ml TGFβ treatment.

Table S1. TUMOR SPECTRUM IN THE Dear1 KNOCKOUT MOUSE MODEL ID Sex Genotype Pathology A47 M Dear1-/- Lung Adenocarcinoma A60 M Dear1-/- High grade Sarcoma A61/OM34 F Dear1-/- Lymphoma A63 F Dear1-/- Lymphoma MID01 M Dear1-/- Lung Adenocarcinoma MID04 F Dear1-/- Lymphoma OM11 F Dear1-/- Lymphoma 378/ F Dear1-/- Lymphoma in liver, stomach, kidney and mammary gland. OM05/331 F Dear1-/- Lymphoma, Basaloid Squamous Carcinoma A57 F Dear1+/- Breast Adenocarcinoma OM07 F Dear1+/- Lymphoma 253/ F Dear1+/- Spindle Cell Sarcoma, low grade 315/318 F Dear1+/- Lymphoma 375/ M Dear1+/- Adenoma/ hyperplasia of small intestine OM07/231 F Dear1+/- Lymphoma 201/ M Dear1+/- Lymphoma 218/ F Dear1+/- Adenocarcinoma, metastatic to the lung. 219/ F Dear1+/- Lacrimal gland adenoma 220/ F Dear1+/- Lymphoma 245/ M Dear1+/- Lymphoma 262/ F Dear1+/- Lung Adenocarcinoma 372 M Dear1+/- Pancreatic Adenocarcinoma, intestinal carcinoma OM35 M Dear1+/- Lymphoma in small intestine

312 M Dear1+/- Hepatocellular carcinoma 337 M Dear1+/- Intestinal carcinoma with liver metastasis

Table S2. DEAR1 Expression in Mouse Tissues

Mouse ID GT Normal Tissue Tumor

OM07 Het Neg Neg

MID01 Het Neg Neg

375 Het Pos Pos A57 Het Neg Neg

218 Het Pos Pos

A47 Het NA** Pos

337 Het Pos Pos

253 Het Pos Neg 245 Het ND* Pos 201 Het Pos Neg 262 Het Pos Neg

315 Het ND Pos 220 Het Pos Pos 378 Het ND Pos

OM35 Het ND Pos 372 Het NA Pos 312 Het Neg Neg 219 Het Pos Pos

*ND: Not determine; **NA: Not available.

Neg: Negative

Pos: Positive.

Supplementary Table S3. DEAR1 is mutated in multiple cancer types.

Exon Mutation Mutation Type

Conservation PolyPhen2 Prediction

Cancer Type Reference

Exon 1 V40M MISSENSE Variable Probably Damaging Upper Aerodigestive Tract Carcinoma

Stransky et al. 2011

Exon 1 Q94K MISSENSE Variable Benign Bladder Carcinoma Gui et al. 2011

Exon 1 D97N

MISSENSE Conserved Possibly Damaging Glioblastoma Multiforme cBIO database (TCGA) (currently not validated by TCGA)

Exon 1 D106V* MISSENSE Conserved Possibly Damaging Breast Adenoca. Exon 2 T167I MISSENSE Conserved Benign Lung Adenoca. Imielinski et al. 2012 Exon 3 R187W MISSENSE Variable Probably Damaging Breast Adenoca. Lott et al. 2009

Exon 3 R187Q MISSENSE Variable Probably Damaging Breast Adenoca. Lott et al. 2009 Exon 3 R190H MISSENSE Variable Probably Damaging Colorectal Adenoca. Cancer Genome Atlas Network 2012 Exon 3 R223H* MISSENSE Variable Probably Damaging Pancreatic Adenoca. Exon 3 R223C MISSENSE Variable Probably Damaging Colorectal Adenoca. Cancer Genome Atlas Network 2012 Exon 3 D240N* MISSENSE Conserved Possibly Damaging Breast Adenoca. Exon 3 R254Q* MISSENSE Conserved Probably Damaging Pancreatic Adenoca. Exon 3 R254Splice

Splice Conserved Probably Damaging Glioblastoma Multiforme cBIO database (TCGA)

(currently not validated by TCGA) Exon 5 R307C MISSENSE Variable Probably Damaging Colorectal Adenoca. Cancer Genome Atlas Network 2012 Exon 5 R307H MISSENSE Variable Probably Damaging Lung Adenoca. Rudin et al. 2012 Exon 5 R333C

MISSENSE Conserved Probably Damaging Endometrial Carcinoma cBIO database (TCGA)

(currently not validated by TCGA) Exon 5 V350I MISSENSE Conserved Probably Damaging Breast Adenoca. Lott et al. 2009 Exon 5 E370K

MISSENSE Conserved Probably Damaging Endometrial Carcinoma cBIO database (TCGA) (currently not

validated by TCGA) Exon 5 D421G MISSENSE Conserved Probably Damaging Colorectal Adenoca. Cancer Genome Atlas Network 2012 Exon 5 Y439H MISSENSE Conserved Probably Damaging Clear Cell Renal

Carcinoma cBIO database (TCGA) (currently not validated by TCGA)

Exon 5 P446S MISSENSE Conserved Probably Damaging Clear Cell Renal Carcinoma

cBIO database (TCGA) (currently not validated by TCGA)

*Identified by our group and report here first. This table documents in detail the mutations of DEAR1 currently discovered. The deleterious degree is predicted by PolyPhen2 (V2.2.2). The conservation is analyzed according to ConSeq (V1.1), and a mutation within conserved region suggests a higher likelihood of being deleterious.

Table S4. DEAR1 exhibits significant copy number loss in a variety of epithelial tumors.

Cancer Type Heterozygous Loss in TCGA

Breast Cancer 33.5% (n=866)

Colorectal Cancer 30.3% (n=575)

Ovarian Serous

Cystadenocarcinoma

24.5% (n=559)

Lung Squamous Cancer 22.9% (n=179)

Kidney Renal Clear Cell

Carcinoma

13.1% (n=436)

Glioblastoma Multifome 8.5% (n=497)

Uterine Corpus Endometriod

Carcinoma

4.7% (n=363)

Table S5. DEAR1 Exhibits Significant Downregulation in a Variety of Tumors.

Cancers that exhibit DEAR1 mRNA downregulation

Median log2 mRNA (Range)

in Normal Samples

Median log2 mRNA (Range)

in Tumor Samples

P-Value

COLORECTAL

Colon Adenocarcinoma (n=101)

.02 (.319 - -.22)

-.433 (-.047 - -.834)

3.09 E-9●

Rectal Adenocarcinoma (n=60)

-.263 (.051- -.593)

-.504 (-.047 - -.942)

7.48 E-10●

Cecum Adenocarcinoma (n=22)

.02 (.319 - -.22)

-.115 (.285 - -.784)

2.98 E-5 Δ

BRAIN Glioblastoma (n=515; n=81)

.305 (.718 - .03); .855 (1.48 -.609)

-.29 (.281 - -

.768);

.52 (1.287 - -

.279)

5.32 E-5 Δ

1.01 E-5 Δ

LYMPHOMA Peripheral T-Cell Lymphoma (n=28)

1.847 (2.511 – 1.08)

.965 (1.448 -.059)

9.58 E-10●

Using data from the TCGA project (colorectal and brain), Sun et al. 2006 (brain n=81), and Piccaluga et al. 2007 (lymphoma), along with analysis from the Oncomine database (oncomine.org), DEAR1 is found to have significant downregulation in multiple tissue types. Significance was assessed using a Bonferroni corrected p-value of 2.45 E-6 for colorectal cancer, 3.96 E-6 (2.55 E-6 for Sun 2006) for brain cancer, and 2.55 E-6 for lymphoma due to multiple comparisons. It is important to note that the data from colorectal cancer and lymphoma also reached a significance level associated with genome wide significance (5 E-8)●. Other data was shown to be approach significance in cecum adenocarcinoma and brain cancer Δ.

Table S6. Correlation between DEAR1 Mutation and Clinical Outcome*

*Cases with Accessible Clinical Information

Mutation Cancer Stage Disease Status Invasive/Metastatic

D106V Breast Carcinoma

N/A N/A Yes; Lymph node involvement

E138K Melanoma N/A Malignant metastatic R187W 21MT cell line;

metastatic breast cancer

IV Cell line was derived from metastaic lesion of patient who had expired from Stage IV breast cancer

Yes; Metastatic

R190H Colon Adenocarcinoma

IIA Recurred/Progressed Invasive

R223C Colon Adenocarcinoma

IIB Recurred/Progressed Invasive

R307C Colon Mucinous I Recurred/Progressed Invasive R333C Endometrial

Carcinoma I; Grade 3

Disease free; Grade 3 indicates poor differentiation/aggressiveness

No

V350I Breast Carcinoma

III N/A Invasive

E370K Endometrial Carcinoma

I; Grade 1

Disease free No

D421G Rectal Carcinoma

IIIC Recurred/Progressed Yes; Lymph node involvement

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2. He W, Dorn DC, Erdjument-Bromage H, Tempst P, Moore MAS, Massague J. Hematopoiesis Controlled by Distinct TIF1β and Smad4 Branches of the TGFβ Pathway. Cell 2006;125:929-41.

3. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data. Cancer Discov 2012;2:401-4.

4. Chen NY, Szentirmay MN, Pawar SA, Sirito M, Wang J, Wang Z, et al. Tumor-suppression function of transcription factor USF2 in prostate carcinogenesis. Oncogene 2006;25:579-87.

5. Stransky et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011 Aug 26;333(6046):1157-60

6. Gui Y, et al. Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder. Nat Genet. 2011;43(9):875-878

7. Cerami et al. The cBio Cancer Genomics Portal: An Open Platform for Exploring Multidimensional Cancer Genomics Data. Cancer Discovery. May 2012 2; 401

8. Imielinski et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell. 2012 Sep 14;150(6):1107-20

9. Lott et al. DEAR1 is a dominant regulator of acinar morphogenesis and an independent predictor of local recurrence-free survival in early-onset breast cancer. PLoS Med. 2009 May 26;6(5)

10. The Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012 Jul 18;487(7407):330-7

11. Rudin et al. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer. Nat Genet. 2012 October; 44(10): 1111–1116.

12. Pier Paolo Piccaluga. PP et al. Gene expression analysis of peripheral T cell lymphoma, unspecified, reveals distinct profiles and new potential therapeutic targets. J Clin Invest. 2007;117(3):823–834.

13. Sun L et al. Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain. Cancer Cell. 2006 Apr;9(4):287-300.

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0.8

0.6

0.4

0.2

WT1 WT2 KO1 KO2

WT Het KO

20

10

30

Surv

ival (

Month

s)

0

C

D

Perc

ent S

urv

ival

Months

Lung Adenocarcinoma Breast Adenocarcinoma

A57(Het)218(Het)E

M200bp

100bp

WT(140bp)

KO(99bp)

N T201

N T245

N T253

N TOM07

(-) (+)F

A B

31 32 33 34 35 36


Urin

.Thyr

d St

om.S

ftT.

Ov

ry

Skin

Panc

Lung

Liv

erKi

dney

Haem

.H&

NGa

stro

.

Bre

ast

Es

oph

CNS

Cerv

ixBo

ne

Cancer Type% of LOH of DEAR1in

CONAN Cell Lines

Bone 15% (n=33)

Breast 18% (n=44)

Cervix 25% (n=12)

CNS 32% (n=96)

Esophageal 23% (n=22)

Gastrointestinal 28% (n=47)

Head & Neck 9% (n=22)

Haematopoietic 7% (n=127)

Kidney 33% (n=21)

Liver 33% (n=9)

Lung 32% (n=151)

Ovary 18% (n=22)

Pancreas 31% (n=16)

Skin 15% (n=47)

Soft Tissue 37% (n=19)

Stomach 19% (n=21)

Thyroid 50% (n=12)

Urinary 22% (n=18)

AdenocarcinomaNormal

AdenocarcinomaAdenocarcinoma


A

B

HP

DE

HP

DE

/KR

as

PA

NC

48

AS

PC

1

MIA

PaC

a2

PA

NC

1

DEAR1

-Actin

1.03 1.04

0.94

0.28 0.290.32 0.32

CshR1 CshR2 CshR3 DshR1 DshR2 DshR3 DshR4

Rela

tive R

NA

exp

ress

ion

(

DE

AR

1/G

AP

DH

) 1.0

0.8

0.6

0.4

0.2


TGF-ß

TGF-ß

TGF-ß

TGF-ß

TGF-ß

TGF-ß

Day 2 Day 5 Day 10 Day 16 Day 20

76N

-E6

Csh

R1

Csh

R2

Dsh

R1

Dsh

R2

Dsh

R3


TG

F

Vector DEAR1

TG

F

CshR DshRB

D


CshR DshRT

GF

A

160

80

40

120

Mig

ratio

n D

ista

nce

(

m/c

ell)

CshR DshR

No TGF

TGF

p<0.001

p<0.001C

CshR DshR(KD2)

Ctr

l

Ctr

l

TG

F

Ctr

l

Ctr

l

E

Rel

ativ

e M

igra

tion

(Fol

d of

Ctr

l)

10A/CshR 10A/DshR

2.0

1.0

1.5

0.5

p<0.001

F

No TGF

TGF

-Integrin

Vimentin

DEAR1

N-Cadherin

E-Cadherin

-Actin

10A/CshR 10A/DshR

TC TC

A

*

*Trace of vimentin after stripping.

TGF

100

60

20

40

80

CshR1 DshR1 DshR2CshR2

B

-Integrin

Vimentin

DEAR1

N-Cadherin

-Actin

CshR DshR(KD2)

TC TC

C

TGF


D

Vim

entin

Posi

tive C

ells

(%

)

Rela

tive E

xpre

ssio

n

(Fold

of C

trl)

400

100

200

300

14

2

6

10

VecDEAR1

SMAD3

400

100

200

300

Vec

A

BCAGA12 PAI-1

Rela

tive L

uci

fera

se A

ctiv

ity (

Fold

of C

trl)

Rela

tive L

uci

fera

se A

ctiv

ity (

Fold

of C

trl)

Vec HA/DR1 Vec HA/DR1

Myc/SMAD3

C

20

10

30

Rela

tive L

uci

fera

se A

ctiv

ity (

Fold

of C

trl)

Vec DEAR1

MDA-MB-231

3

1

2

4

5

Vec DEAR1

HeLa

No TGF

TGF

No TGF

TGF

No TGF

TGF

No TGF

TGF

No TGF

TGF


Vec HA/DR1 Vec HA/DR1

Myc/SMAD3

D


SMAD3

-Actin

CshR DshR(KD2)

TC TCTGF

A

SMAD3

DEAR1

Coom

asi

e B

lue

NT N TTN TN TT N TN

BC1 BC2 BC4 BC5 BC7BC6

N

BC3

T/N

T/N

T

um

or/

Norm

al

(Norm

aliz

ed to P

rote

in)

3

2

SMAD3

DEAR1

BC1 BC2 BC4 BC5 BC7BC6BC3

Breast Cancer Samples

1SMAD2

HA/DEAR1 Flag/SM3 Nuclei Merge

SM

AD

3/N

SM

AD

3/L

&C

IP:anti-HA

Input

HA(DEAR1)

Flag(SM3)

Flag(SM3/LC)

Flag(SM3/N)

Vect

or

SM

3/N

SM

3/L

C

HA(DEAR1)B

CHA(DEAR1)

SMAD3DEAR1

a

c

bb

d

f

be


B C


P-S

MA

D3

Nu

cle

iM

erg

eCshR-NT CshR-TGF DshR-NT DshR-TGFA

CshR1 CshR2 DshR1 DshR2

Ctr

lT

GF

0 0 1 2 5SIS3 (uM)

CshR

0 0 1 2 5

DshR

- + + + +TGF - + + + +

Vimentin

N-Cadherin

-Actin

SMAD3

Fibronectin

MCF10AA

B

TG

F

SIS

3


dear1 and master regulator of tgfβ-driven epithelial ... · e6 cells) (dshr) as compared to...

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