G. Travis Tabor, Darren F. Seals†, Jennifer P. Cecile‡

Departments of Biology† and Chemistry‡

Appalachian State University, Boone NC 28608

Actin ECMInvadopodia

Exploring Possible Roles of Organic Anion-Transporting Polypeptides in

Invadopodia Function

2

• Drug resistant, invasive tumors are among the most devastating cancers. It is the goal of this project to explore the potential relationships between these two phenotypes in a variety of tissue culture model systems.

• Invadopodia are actin-rich protrusions of the cellular membrane that allow cancer cells to invade other tissues during metastasis.

• Organic anion transporting-polypeptides (OATPs) are proteins that mediate xenobiotic exchange across the cellular membrane.

• Invadopodia and OATPs are believed to be associated with lipid raft domains (LRDs) and caveolin-1 in humans.

• To determine if functional OATPs are present in invadopodia-competent cells, fluorescence transport assays were performed on a variety of cancer cell lines.

• All cell lines exhibited robust uptake of rhodamine-123 (Rh-123), a fluorescent substrate of OATP1A2, and an inhibition assay indicated this transporter may be responsible for the observed uptake of Rh-123 by LNCaP cells grown in androgen-depleted conditions.

• If OATPs play a role in invadopodia activity, they may contribute to the enhanced invasive phenotype of certain cancers and therefore serve as viable therapeutic targets.

Abstract

3

Invadopodia

A B

• Actin-dense, lipid-rich ventral protrusions of the cell membrane1

• Exert motive force on and mediate remodeling of ECM

• Putatively involved in the metastasis of certain cancers

• MT1-MMP, lipid rafts, and caveolin-1 are critical for proper functioning in breast cancer and melanoma cells3-6

Figure 1. Invadopodia structure and function in Src-3T3 cells. (A) Invadopodia (green arrow) form rosette superstructures (white arrow) in mature, invasive cancer cells. Actin was stained with phalloidin 594 and nuclei with DAPI. (B) Black areas correspond to invadopodia-mediated matrix degradation. Coverslips were coated with Alexa Fluor 488-conjugated gelatin to simulate extracellular matrix (ECM), and nuclei were stained with DAPI.

4

• 12-transmembrane glycoproteins that mediate the transfer of large hydrophobic organic anions such as steroid hormone conjugates, bile acids, and toxins7

• Believed to play a role in multi-drug resistance and thus contribute to the malignancy of certain cancers8

• Organic anion transporters (OATs) are localized to lipid-rafts and co-expressed with caveolin 19

• Shown to be upregulated in androgen-starved LNCaP cells10

OATPs

Figure 2. Fold recognition model of OATP1B1 and OAT1.3 OATPs and OATs are homologous proteins that contain 12 transmembrane helical domains. (Right) OATP1B1 and (Left) OAT1 models were generated using Phyre, a protein homology recognition server.

5

• Castration resistant prostate cancer is significantly more difficult to treat than the androgen-dependent form11

• Androgen receptor (AR) regulates cell growth and motility10,12

• OATP1A2 transports DHEAS, an inactive androgen precursor produced by adrenal glands10

• DHEAS is converted to androstenedione which eventually activates AR and stimulates cell growth10,13

OATPs, AR, and Castration Resistance

Figure 3. DHEAS metabolism and prostate cancer interventions.13

6

• Src was the first discovered oncogene and induces invadopodia development when overexpressed in NIH-3T314

• Src is believed to potentiate AR activity in the absence of gonadal androgens (i.e. in castration resistant prostate cancer)15

• AR activates Src in PC3 cells overexpressing AR, stimulates filopodia formation and migration16

• Stimulation of AR by R1881 induced rapid formation of cytoskeletal ruffles and protrusions and enhanced motility in NIH-3T3 cells12

Src, AR, and Castration Resistance

Figure 4. Roles of Src in invadopodia formation.17

7

Proposed OATP1A2-AR-Src Signaling

Src

A

AR

Src

Cleavage,processing

Invadopodia regulatorsActivity

Cell division,motility

OATPAP

AP

A = Active Androgen

= Androgen Receptor

= Androgen PrecursorAP

Figure 5. Proposed mechanism for OATP-AR-Src signaling in invadopodia formation and development.

+ OATP substrate dyes30 m, 37°C, dark

Grow cells for 18 hours in 6-well dishes

Wash with cold HBSS

+ 1 M NaOH, scrapeLyse 1 hour, 37°C, dark

Neutralize with 6 M HCl

Bradford AssayFluorescence Assay

Excite at 485 nmMeasure at 535 nm

Normalize fluorescence intensity to protein concentration

Initial Fluorescence Transport Assay

Microplate reader image from www.news-medical.net8

Figure 6. Initial Fluorescence Transport Assay Protocol.

9

DyeMaximum Wavelength (nm)

Target(s)7 Km (µM)18,19

Excitation Emission

Rhodamine 506 529 OATP1A2 0.3

Fluorescein-methotrexate (FMTX)

496 516OATP1B1 5.2

OATP1B3 8.9

Fluo-3 pentapotassium salt 506 526 OATP1B3 2.3

Fluo-3 AM (+) 488 525 Permissible N/A

Rhodamine-123 FMTX

Fluo-3 AMFluo-3

Materials

Chemical and structures and wavelength data from ThermoFisher Scientific

Cell line Tumor type

MDA-MB-231 Human breast

LNCaP Human prostate

PC3 Human prostate

Src-3T3 Murine fibroblast

Fluo-3 AM (+)

Rh-123

FMTX Fluo-3 Rh-123

FMTX Fluo-3 Rh-123

FMTX Fluo-3

Src-3T3 PC3 MDA-MB-231

0

10

20

30

40

50Fo

ld B

ackg

rou

nd

Nor

mal

ized

Flu

-or

esce

nce

Inte

nsi

ty (

535

nm

)

Figure 7. Uptake of OATP substrate dyes by Src-3T3, PC3, and MDA-MB-231 cell lines. Data are the fold background normalized fluorescence intensities at 535 nm from a representative experiment. Each cell line tested took up rhodamine-123 (Rh-123), a fluorescent substrate of OATP1A2. Transport of FMTX and fluo-3 was significantly lower than that of rhodamine-123.

OATP Fluorescent Substrate Uptake Screen

10

11

Figure 8. Uptake of selected OATP substrate dyes by Src-3T3, PC3, and MDA-MB-231 cell lines. Further replicates are required to determine if the signal from the FMTX and fluo-3 samples are significantly different from that of the background.

Fluo

-3 A

M (

+)

FMTX

Fluo

-3

FMTX

Fluo

-3

FMTX

Fluo

-3

Src-3T3 PC3 MDA-MB-231

0

0.5

1

1.5

2

2.5Fo

ld B

ackg

rou

nd

Nor

mal

ized

Flu

o-re

scen

ce In

ten

sity

(53

5 n

m)

OATP Fluorescent Substrate Uptake Screen

12

37°C 0°C0

2

4

6

8

10

12

14

16

18

20

Incubation temperature

Fold

Bac

kgro

un

d N

orm

aliz

ed F

lu-

ores

cen

ce In

ten

sity

(53

5 n

m)

Figure 9. Effect of temperature on uptake of rhodamine-123 by MDA-MB-231 cells. Uptake of rhodamine-123 by MDA-MB-231 cells was significantly reduced under cold conditions. The fluorescence intensity at 535 nm of the cold treatment lysate was 67% less than that of the 37°C treatment lysate.

Initial Ice Inhibition Experiment

13

FTA Precision Issue?

37°C 0°C02468

101214161820

Incubation temperature

Fold

Bac

kgr

oun

d N

orm

aliz

ed

Flu

ores

cen

ce I

nte

nsi

ty (

53

5 n

m)

Fluo

-3 A

M (

+)

Rh-

123

FMT

X

Fluo

-3

Rh-

123

FMT

X

Fluo

-3

Rh-

123

FMT

X

Fluo

-3

Src-3T3 PC3 MDA-MB-231

0

10

20

30

40

50

Fold

Bac

kgr

oun

d N

orm

aliz

ed

Flu

ores

cen

ce I

nte

nsi

ty (

53

5 n

m)

While the general trends were informative, these experiments revealed a potential problem with assay precision

pH effects on Rh-123 fluorescence?20

14

0 1 2 3 4 5 60

5000

10000

15000

20000

25000

30000

35000

40000

45000

HBSS

1% Triton X-100/HBSS

Rhodamine-123 Concentration (µM)

Flu

ores

cen

ce U

nit

s (x

104

)

Figure 10. Fluorescence transport assay lysis buffer analysis. The presence of 1% Triton X-100 detergent reduced rhodamine-123 fluorescence compared to that in empty buffer at high dye concentrations.

Assay Optimization: Buffered Lysis System

15

Optimized Fluorescence Transport Assay+ OATP substrate dyes

5 m, 37°C, dark

Grow cells for 18 hours in 6-well dishes

Wash with cold HBSS

+ 500 µL 1% Triton/HBSS, scrape,vortex, lyse 15 m at 0°C in dark,

centrifuge 10 m at 10k x g

Fluorescence Assay

Excite at 485 nmMeasure at 535 nm

Figure 11. Optimized Fluorescence Transport Assay Protocol.

16

OATP Drug Inhibition Assays

Inhibitor Target7,21

Sodium taurocholate OATP1A2, OATP1B1, OATP1B3, OAT3

Rifampin OATP1A2, OATP1B1, OATP1B3

Estrone-3-Sulfate OATP1A2, OATP1B1, OATP1B3, OAPT1C1

Ketoconazole OATP1A2, OATP1B1, OAT1

Initial Protocol

Inhibitors were simultaneously applied with dye during incubation step

Updated Protocol

Cells were pre-incubated with inhibitor for 10-30 minutes before applying dye

17

Rh-123 Only Rh-123 Only Rifampin Taurocholate0℃ 37℃

0

20

40

60

80

100

120Fo

ld B

ackg

rou

nd

Flu

-or

esce

nce

Inte

nsi

ty

Figure 12. Effects of OATP inhibitors on long-term rhodamine-123 uptake by PC3 cells. Rhodamine-123 (Rh-123) uptake by PC3 cells was not significantly inhibited by rifampin (0.1 mM) or sodium taurocholate (0.3 mM) when incubated for 30 minutes at 37°C. Furthermore, it appeared that sodium taurocholate may have actually inhibited dye export. Also shown is the inhibition of rhodamine-123 uptake by PC3 cells in cold conditions. Here the fluorescence signal of the ice treatment lysate was 57% lower than that of the 37°C condition. Standard deviations from 3 technical replicates per treatment are shown from a representative experiment.

OATP Inhibition in PC3 (30 m)

18

Time Course of Rh-123 Uptake by PC3 Cells

0 1 2 3 4 5 6 7 8 9 100

2

4

6

8

10

12

Incubation Time (m)

Fold

Bac

kgro

un

d F

luo-

resc

ence

Inte

nsi

ty

Figure 13. Time course of rhodamine-123 uptake by PC3 cells. PC3 cells were demonstrated to take up rhodamine-123 rapidly within the first 5 minutes of incubation. The decreasing slope of the curve indicated intracellular rhodamine-123 may reach its equilibrium concentration after 30 minutes. Standard deviations from 3 replicates of each concentration are shown. (Optimized assay)

19

OATP Inhibition in PC3 (5 m)

Rh-123 Only Rifampin Taurocholate E3S0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0Fo

ld B

ackg

rou

nd

Flu

-or

esce

nce

Inte

nsi

ty

Figure 14. Effects of OATP inhibitors on early rhodamine-123 uptake by PC3 cells. Rhodamine-123 uptake by PC3 cells was not significantly inhibited by rifampin (0.1 mM), sodium taurocholate (0.3 mM), or estrone-3-sulfate (E3S, 0.3 mM) when incubated for 5 minutes at 37°C. Conversely, both taurocholate and E3S where shown to increase the amount of intracellular rhodamine-123. Also shown are standard deviations derived from 3 technical replicates per treatment from a representative experiment.

20

Figure 15. Effects of OATP inhibitors on early rhodamine-123 uptake by LNCaP cells grown in androgen depleted conditions. Only ketoconazole was shown to significantly inhibit rhodamine-123 uptake by LNCaP cells grown in androgen-depleted conditions as the fold fluorescence intensity of the cell lysate was 50.3% less than that of the Rh-123 only treatment.

OATP Inhibition in Quiescent LNCaP Cells

Rh-123 Only

Ri-fampi

n

Tau-ro-

cholate

E3S Ke-to-

conazole

0

2

4

6

8

10

12

Fold

Bac

kgro

un

d F

luo-

resc

ence

Inte

nsi

ty

21

Results Summary1. A Rh-123-based transport assay was optimized for mammalian cancer

cell lines

2. Src-3T3, PC3, MDA-MB-231, and LNCaP cell lines exhibited robust uptake of Rh-123 dye while FMTX and fluo-3 uptake was relatively minimal

3. Rh-123 uptake by MDA-MB-231 and PC3 cells was significantly reduced in cold conditions

4. Uptake of Rh-123 by LNCaP cells grown in androgen-depleted conditions was significantly inhibited by ketoconazole, an OATP1A2 inhibitor

22

DiscussionOATP Identification

• OATP1A2 is not likely responsible for all of the observed Rh-123 uptake by MDA-MB-231 cells as this line expresses OATP1A2 mRNA at low levels22

• Inhibition of Rh-123 uptake by LNCaP cells grown in androgen-depleted conditions with ketoconazole indicated endogenous OATP1A2 activity

• However, E3S and taurocholate did not significantly inhibit uptake which countered the results of Arakawa et al. who used radiolabeled DHEAS10

Ketoconazole

• Known to inhibit OATP1A2 import and p-glycoprotein export23,24

• Also used to treat castration-resistant prostate cancer by inhibiting androgen biosynthesis24,25

• Ketoconazole/Rh-123 fluorescence transport assay system may be useful for assessing OATP1A2 activity

23

DiscussionRhodamine-123 Considerations

• First believed to be a membrane-permeable dye19

• Later, uptake was found to be primarily mediated by OATP1A2 below micelle critical limit (2 µM)

• Characterized as a p-glycoprotein export tracer

• Carcinoma cells take up more Rh-123 than the corresponding normal tissue26

Inherent Challenges of Fluorescence Transport Assays

1. OATPs transport many substrates and have overlapping affinities

2. OATPs have differential affinities for substrates and inhibitors

3. Substrates have differential affinities for importers and exporters

Ideal Experimental System

• Radiolabeled substrates

• Use primary tissue or overexpress transporters in cell line

24

1. Continue to perform inhibitor assays to confirm identities of functional transporters

2. Attempt to determine localization and protein expression levels of endogenous OATPs/OATs via fluorescence microscopy and Western blotting

3. Assess OATP/OAT localization during invadopodia development by overexpressing these transporters with Src and Tks5 in a variety of cell lines

4. Determine the effects of OATP inhibition on in situ matrix degradation and invadopodia morphology

5. Determine effects of AR stimulation and inhibition on invadopodia activity and development

6. Explore OAT/OATP function in vivo using a C. elegans anchor cell/invadopodia model27

Future Work

25

Proposed OATP1A2-AR-Src Signaling

Src

A

AR

Src

Cleavage,processing

Invadopodia regulatorsActivity

Cell division,motility

OATPAP

AP

A = Active Androgen

= Androgen Receptor

= Androgen PrecursorAP

Figure 5. Proposed mechanism for OATP-AR-Src signaling in invadopodia formation and development.

Gelatin Degradation Assay OverviewHarvest cells during

log growth phase

Incubate cells for 24-48 hours at 37°C in 12-well dish containing gelatin-coated coverslips

Harvest slipsFix, permeabilize, stain

Mount onto slides

Visualize using fluorescence microscopy

Resuspend in media containing OATP inhibitor

Normalize gelatin degradation to number of nuclei using Image J software

Microscope image from www.microscope.com26

Figure 16. Gelatin Degradation-OATP Inhibition Assay Overview

27

Conclusions• OATs (and assumedly OATPs) may be localized to invadopodia as both structures are associated with lipid rafts and caveolin-1.

• In order to determine if OATPs contribute to invadopodia activity, a series of inhibition assays were performed.

• It is possible that OATPs (or similar drug transporters) are present in the invadopodia-competent cancer cells used in the present study.

• Rh-123 uptake by LNCaP cells grown in androgen-depleted conditions is likely mediated by OATP1A2.

• In the future, the localization and expression of OATPs will be determined using fluorescence microscopy and Western blotting respectively.

• If OATPs are found to localize to invadopodia and contribute to both the drug-resistant and invasive phenotypes of aggressive cancer cells, they may be effective targets for therapy in the future.

28

Acknowledgements• ASU Office of Student Research for multiple Undergraduate Research

Assistantships, research awards, and travel grants

• ASU Honors College for a Partnership Board Research grant that was used to purchase most of the dyes and inhibitors used in the present study

• Amazing mentors: Drs. James, Cartaya, Babyak, Jones, and Celeste Crowe

• Research advisors: Drs. Jennifer Cecile and Darren Seals

• My mom, Martha Brophy, and my brother, Trent Tabor

In memory of my father, George Roley Tabor

29

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required for podosome formation and function, and for the protease driven invasion of cancer cells. Cancer Cell 2005, 7(2), 155-165.

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