Slides from a PowerPoint presentation given by Jackson David Reynolds on Monday, March 28, 2016 at the University of North Georgia, Dahlonega campus for Senior Seminar in Biology (professor: Dr. Ryan Shanks, PhD).

1. Jackson David Reynolds
University 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

2. 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

3. Cytoplasm Nucleus
KSHV brings about angiogenesis via activation
of pro-inflammatory cascades.
Chang et al., 2016. Figure modified from:Chang et al., 2016.
NOTTOSCALE
= miR-221/-222 promoter
= cytokine transcripts
Kaposin
B
c-myc
KSHV
∴ ↑[cytokines]cytosolANGIOGENESIS
3
= miR-221/-222

4. How do we know this?
4 Figures (right to left) from:Lavigne et al., 1998;Kelley LA et al., 2015.

5. HMEC1 migration was assessed with a
Transwell® migration assay.
HMEC1 cells
Microporous
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
NOTTOSCALE
Lower
compartment

6. HMEC1 migration was assessed with a
Transwell® migration assay.
Chang et al., 2016. Main figure modified from:Corning Inc. Life Sciences,August, 2014. Figure of VEGF-A structure:Eww, 2009.
HMEC1 cells
Microporous
membrane
Transwell®
insert Upper
compartment
VEGF-A
Post-incubation
Migrated KSHV+ & CTRL cells
= NML
= KSHV-infected
NOTTOSCALE
6
Lower
compartment

7. 1
1.83
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
EC (NML) Kaposin B
NumberofMigratedCells
Cell Migration
HMEC1 cells overexpressing Kaposin B
migrated 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

8. Kaposin B expression encourages
rapid 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

9. Okay, great – but what about c-myc?
Figure from:Lavigne et al., 1998.9

10. c-myc seemed a likely candidate for
Kaposin 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

11. 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
NOTTOSCALE

12. 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

13. 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

14. c-myc was detected via IP of anti-FLAG-tagged mAb.
Kaposin B DYKDDDDK
Anti-FLAG-
tagged
mAb
14 Chang et al., 2016. Figure created by presentation author.
c-mycYPYDVPDYA
NOTTOSCALE

15. And Kaposin B was detected via IP of anti-HA mAb!
Chang et al., 2016. Figure created by presentation author.
c-myc
Anti-HA
mAb
YPYDVPDYA
15
KaposinBDYKDDDDK
NOTTOSCALE

16. Okay, great – but
maybe c-myc is sufficient rather than necessary
to induce pronounced angiogenesis?
Figure from:Lavigne et al., 1998.16

17. 1
2
0.96
0
0.5
1
1.5
2
2.5
Tubuleformation(%)
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

18. 1 1.89 0.81
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
CTRL KB+/c-myc+ c-myc knockdown
NumberofMigratedCells
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

19. So, what’s next?
19 Figures (top to bottom) from:Lavigne et al., 1998;Kelley LA et al., 2015.

20. Antiangiogenic Rx Tx could potentially
circumvent this cascade.
Kanno et al., 2015. Figure from:Charlesy, 2012.
■ Fumagillin (shown right).
■ Activates lytic cycle in latent
KSHV+ cells.
– ReplicationTranscription
Activator (RTA) expression.
■ ∴ Latent à Lytic.
20

21. Cytoplasm Nucleus
KSHV brings about angiogenesis via activation
of pro-inflammatory cascades.
Chang et al., 2016. Figure modified from:Chang et al., 2016.
NOTTOSCALE
= miR-221/-222 promoter
= cytokine transcripts
Kaposin
B
c-myc
KSHV
∴ ↑[cytokines]cytosolANGIOGENESIS
21
= miR-221/-222

22. Questions?
Figure from:Lavigne et al., 1998.22