Jackson David ReynoldsUniversity of North Georgia

Kaposin B Interacts with c-myc to Engender Angiogenesis in Kaposi Sarcoma Neoplasms

Figures (right to left) from: Lavigne et al,1998; Kelley LA et al., 2015; University of North Georgia, 2013.1

Kaposi Sarcoma is an oncoviral pathology.■ Human Herpesvirus type 8 (HHV-8).– Kaposi Sarcoma Associated

Herpesvirus (KSHV).

■ Immunodeficiency-associated.

■ Tumors characterized by angiogenesis.

American Cancer Society, Inc., 2016. Figures (clockwise) from: National Cancer Institute, 2001;Pinto-Almeida et al., 2011; Silverman, Jr. & Centers for Disease Control and Prevention Public Health Image Library (PHIL), 1999; Henderson & Physicians Research Network (PRN®), 2002.2

Cytoplasm Nucleus

KSHV brings about angiogenesis via activationof pro-inflammatory cascades.

Chang et al., 2016. Figure modified from: Chang et al., 2016.

NO

T T

O S

CA

LE

= miR-221/-222 promoter

= cytokine transcripts

Kaposin B

c-myc

KSHV

∴ ↑[cytokines]cytosolANGIOGENESIS

3

= miR-221/-222

How do we know this?

4 Figures (right to left) from: Lavigne et al., 1998; Kelley LA et al., 2015.

HMEC1 migration was assessed with aTranswell® migration assay.

HMEC1 cellsMicroporous membrane

Transwell®

insert Upper compartment

Vascular endothelial

growth factor A (VEGF-A)

Chang et al., 2016. Main figure modified from: Corning Inc. Life Sciences, August, 2014. Figure of VEGF-A structure: Eww, 2009.

= NML

= KSHV-infected

5

NO

T TO

SC

ALE

Lowercompartment

HMEC1 migration was assessed with aTranswell® migration assay.

Chang et al., 2016. Main figure modified from: Corning Inc. Life Sciences, August, 2014. Figure of VEGF-A structure: Eww, 2009.

HMEC1 cellsMicroporous membrane

Transwell®

insert Upper compartment

VEGF-A

Post-incubation

Migrated KSHV+ & CTRL cells

= NML

= KSHV-infected

NO

T TO

SC

ALE

6

Lowercompartment

1

1.83

00.20.40.60.8

11.21.41.61.8

2

EC (NML) Kaposin B

Num

ber

of M

igra

ted

Cel

ls

Cell Migration

HMEC1 cells overexpressing Kaposin Bmigrated more than uninfected cells.

Chang et al., 2016. Figure modified from: Chang et al., 2016.

■ n = 3 (per trial).

■ Confirmed previous findings:

– Kaposin B positively regulates

“angiogenesis-like” motility.

7

Kaposin B expression encouragesrapid tubule formation in vitro.

Chang et al., 2016. Figures modified from: Chang et al., 2016.

■ KSHV+ HUVECs & CTRL on

simulated basement membrane.

■ 96% increase in modeled

microvasculature formation.

Tubule formation

KSHV+KSHV-

8

Okay, great – but what about c-myc?

Figure from: Lavigne et al., 1998.9

c-myc seemed a likely candidate forKaposin B interaction.

Chang et al., 2016. Main figure modified from: Chang et al., 2016. c-myc figure (left) from: Lavigne et al., 1998.

■ Three putative binding sites of c-myc

on the miR-221/-222 promoter

previously identified:

1. E1

2. E2

3. E3

10

Kaposin B/c-myc interaction was assayed via co-transfection of HEK 293 cells and subsequent IP.

Chang et al., 2016. Figure created by presentation author.

Human Embryonic

Kidney 293 cell

Kaposin B DYKDDDDK

c-myc YPYDVPDYA

Nucleus

FLAG-tag

HA tag

Products of co-transfected DNA

(from KSHV)

11

NO

T TO

SC

ALE

Immunoprecipitation demonstrated protein-protein interaction.

Promega, 2013.12

Detect protein 2 via gel electrophoresis

Immunoprecipitate w/antibody against protein 1

MIX

Kaposin B gene product c-myc gene product

Immunoprecipitation demonstrated protein-protein interaction.

Promega, 2013.13

Detect protein 1 via gel electrophoresis

Immunoprecipitate w/antibody against protein 2

MIX

Kaposin B gene product c-myc gene product

c-myc was detected via IP of anti-FLAG-tagged mAb.

Kaposin B DYKDDDDKAnti-FLAG-

taggedmAb

14 Chang et al., 2016. Figure created by presentation author.

c-myc

YPY

DV

PDYA

NO

T TO

SC

ALE

And Kaposin B was detected via IP of anti-HA mAb!

Chang et al., 2016. Figure created by presentation author.

c-myc Anti-HAmAb

YPYDVPDYA

15

Kaposin B

DY

KD

DD

DK

NO

T TO

SC

ALE

Okay, great – but

maybe c-myc is sufficient rather than necessary

to induce pronounced angiogenesis?

Figure from: Lavigne et al., 1998.16

1

2

0.96

0

0.5

1

1.5

2

2.5

Tubu

le fo

rmat

ion

(%)

Tubule-Modeled “Angiogenesis”

c-myc knockdown severely retarded modeled angiogenesis in Kaposin B-expressing HUVECs.

Chang et al., 2016. Figure modified from: Chang et al., 2016.

CTRL(Kaposin B-/c-myc+)

c-myc+;Kaposin B+

c-myc knockdown(Kaposin B+)

17

1 1.89 0.81

00.20.40.60.8

11.21.41.61.8

2

CTRL KB+/c-myc+ c-myc knockdown

Num

ber

of M

igra

ted

Cel

ls

Migration-Modeled “Angiogenesis”

c-myc knockdown severely retarded modeled angiogenesis in Kaposin B-expressing HUVECs.

Chang et al., 2016. Figure modified from: Chang et al., 2016.18

So, what’s next?

19 Figures (top to bottom) from: Lavigne et al., 1998; Kelley LA et al., 2015.

Antiangiogenic Rx Tx could potentiallycircumvent this cascade.

Kanno et al., 2015. Figure from: Charlesy, 2012.

■ Fumagillin (shown right).

■ Activates lytic cycle in latent

KSHV+ cells.

– Replication Transcription

Activator (RTA) expression.

■ ∴ Latent à Lytic.

20

Cytoplasm Nucleus

KSHV brings about angiogenesis via activationof pro-inflammatory cascades.

Chang et al., 2016. Figure modified from: Chang et al., 2016.

NO

T T

O S

CA

LE

= miR-221/-222 promoter

= cytokine transcripts

Kaposin B

c-myc

KSHV

∴ ↑[cytokines]cytosolANGIOGENESIS

21

= miR-221/-222

Questions?

Figure from: Lavigne et al., 1998.22