Polo-Like Kinase 1 in Genomic Instability

Polo-like Kinase 1 in genomic instability

Xiaoqi Liu

Department of Toxicology and Cancer Biology

University of Kentucky The fate of a single parental chromosome throughout the eukaryotic cell cycle Cytokinesis Activation of cell division

Preparation for mitosis The stages of mitosis and cytokinesis in an animal cell

MT MT - + kinetochore Centrosome Spindle pole + (MTOC) Spindle pole Centrosome Spindle

Nucleus

G2 Prophase Prometaphase Metaphase

Central spindle Contractile ring Midbody

Anaphase Telophase Cytokinesis Control of mitotic entry and exit by Cdc2/cyclin B and APC

Anaphase promoting complex (APC) Cdc2 Cyclin B

Cdc2 Anaphase inhibitor Cyclin B Cdc2

G2 Prophase Metaphase Anaphase Telophase G1

(Liu & Erikson, PNAS 2002; Liu & Erikson, PNAS 2003; Liu et al, MCB 2005; Liu et al, MCB 2006) The polo-like kinases have a C-terminal polo-box domain M-phase kinase domain polo-box Plk1 603

Plx1 (Xl) 80% 598

polo (Dm) 65% 576

Plo1 (Sp) 50% 683

Cdc5 (Sc) 51% 705

Early phase of cell cycle

Plk2 52% 683

Plk3 52% 631 Plk1 localizes to mitotic structures G2/pro Prometa

Spindle poles Centrosomes kinetochores

Centrosomes Centrosomes kinetochores

Meta Telo

Spindle poles Midbodies kinetochores

Spindle poles Central spindles kinetochores Plk1 in cancer

---Plk1 overexpression is correlated with cell proliferation and carcinogenesis.

---Plk1 is a new diagnostic marker for cancer.

---Plk1 inhibitors are in various clinical trails.

---However, how Plk1 contributes to carcinogenesis is elusive. Does Plk1 elevation contribute to cancer progression?

Approach: generation of Plk1-Knock in (KI) mouse line Plk1 KI mice Phenotypes

Plk1 KI causes aneuploidy in MEFs No apparent phenotypes for Plk1-KI mice, but they are hyper-sensitive to IR

DDB

ATM

Chk2 gH2AX

p53

Cell cycle arrest & DNA repair A liver B Ctrl CMV-Cre/Plk1-KI IR-treated, liver

CMV-Cre/Plk1-KI H&E 50µm

Plk1-KI mice have

Plk1 Plk1 -

increased incidence anti 50µm

of lymphoma or Ki67 Ki67

- Ctrl

severe fatty change anti 50µm

IR-treated, liver T cell lymphoma

upon IR C D anti-CD3

/Plk1

Ctrl Cre Multifocal lymphoid hyperplasia - in perivascular location CMV 200µm

200µm 100µm 50µm

KI -

Mild lymphoma -

/Plk1

Cre

Cre -

Multiple lymphocytic - CMV CMV 100µm

overgrowth in the parenchyma

/Plk1

/Plk1 Cre

Severe lymphoma -

Cre

- CMV

CMV 50µm

E Ctrl CMV-Cre/Plk1-KI CMV-Cre/Plk1-KI F Fatty liver

Ki67

/Plk1

Cre

anti -

200µm 200µm 100µm 50µm CMV 50µm Plk1 KI inhibits expression of DNA damage repair genes

Dr. Zhiguo Li Dr. Jinghui Liu More specific mechanism?

DNA double strand breaks

MRN complex (Mre11/Rad50/Nbs1) Plk1

ATM

Chk2

p53

Cell cycle arrest DNA repair A hypothesis to be tested

Plk1 elevation leads to

1) premature termination of DNA damage checkpoint and

2) reduced DNA repair Plk1-associated activity antagonizes DNA damage checkpoint Plk1 phosphorylates Mre11 at S649 and S688 in vitro In vivo, Plk1 phosphorylates Mre11-S649 and CK2 targets Mre11-S688 Mre11-S649/S688 phosphorylation is required for G2 DNA damage checkpoint recovery

1st T block 8h rel. 2nd T block 7h rel. Dox for 1h caffeine for 3h p-H3 IF Phosphorylation of Mre11 at S649/S688 inhibits its binding to DNA

Xenopus egg extract + Purified Mre11 proteins + Biotin-tagged dsDNA-bound avidin beads

Pellets

Mre11 IB Phosphorylation of Mre11 at S649/S688 abolishes its foci formation Phosphorylation of Mre11 at S649/S688 abolishes Nbs1 foci formation Phosphorylation of Mre11 at S649/S688 abolishes Rad50 foci formation Phosphorylation of Mre11 at S649/S688 inhibits DNA damage checkpoint Mre11-S649/S688 phosphorylation inhibits DNA repair

A B Conclusions

Plk1 phosphorylation of Mre11 leads to

1) premature termination of DNA damage checkpoint

2) reduced DNA repair

Publications: Li et al., Cancer Research, 2017, 77, 3169.

Li et al., JBC, 2017, 292, 17461. Dr. Zhiguo Li MDC1 • MEDIATOR OF DNA DAMAGE CHECKPOINT

DNA damage MRN (Mre11/Rad50/Nbs1)

Initiation of damage response Plk1

MDC1 Amplification of damage response

Bekker-Jensen and Mailand, DNA repair, 2010, 9, 1219 MDC1 KO phenotype

MDC1 heterozygous mice are cancer prone by a DDR-independent pathway MDC1 reduction causes aneuploidy A

Foci formation in regular dish

MDC1 heterozygosity sensitizes transformation of immortalized MEFs

Soft agar assay

To immortalize, MEFs were transfected with SV40 large T antigen MDC1 is required for mitotic progression Double Thymidine Block--- Release

G1 MDC1 is required for timely progression of mitosis

HeLa expressing GFP-H2B

HeLa expressing GFP-a-tubulin MDC1 is phosphorylated in mitosis Plk1 phosphorylates MDC1 at T4 p-T4-MDC1 localization in prophase and prometaphase

Nuclear envelope Centrosomes

Kinetochores Centrosomes p-T4-MDC1 localization in mitosis

Kinetochores

Microtubule (+) ends

Midbody p-T4-MDC1 localization in mitosis

Nuclear envelope

Kinetochores

Midzone

Midbody Plk1 phosphorylation of MDC1-T4 is required for mitotic progression Double Thymidine Block --- release

G1 Noc release

G1 Plk1 phosphorylationPlk1 phosphorylatesof MDC1-T4 is required MDC1 at for T4 mitotic progression Plk1 phosphorylatesSummary MDC1 at T4

--MDC1 has a function independent of DNA damage response

--MDC1 is required for mitosis

--MDC1 is phosphorylated by Plk1 at T4

--Plk1 phosphorylation of MDC1-T4 is required for timely progression through mitosis

Publications: Li et al., Mol Cell Biol, 2017, 37, e00595.

Dr. Zhiguo Li PThelk1 phosphorylates Numb/p53 MDC1pathway at T4

Numb

MDM2 p53

-- Numb is an inhibitor of Notch signaling

-- Numb is involved in the cell-fate decisions of a number of cell lineages

-- Numb, MDM2 and p53 form a trimeric complex

-- Numb stabilizes p53 by inhibiting the E3 ubiquitin ligase activity of MDM2

Colaluca, Nature 2008, 451, 76-80. Plk1 phosphorylates Numb at S265 and S284 in vitro

GST-Numb (aa) GST-Numb (aa)

GST-Numb (aa) Numb-S265 is phosphorylated in vivo in a Plk1 dependent manner Numb degradation is enhanced by Plk1-associated kinase activity Plk1 phosphorylation of Numb results in its proteasome degradation Plk1 negatively regulates the Numb/p53 pathway

* Plk1-KI MEFs * Tamoxifen - + * DOX + + 24 * Plk1 22 20 * p53 * 18 p21 16 g-H2AX 14 β-actin 12 c 10 8 A higher binding affinity between p53 and Numb-AA p53 reporter RLU 6 Flag-Numb WT AA DD 4 2 Flag 0

p53

β-actin p53/actin 1.0 3.3 1.2

Flag IP:Flag GFPGFP--Plk1 p53 FlagFLAG β-actin U2OS cells β-actin Plk1 phosphorylation of Numb contributes to p53 degradation

Numb WT AA DD

180KD IP:p53 IB:Ub

90KD

p53

FLAG p53 β-actin

p53/β-actin 1 2.7 1.6 Cells expressing Numb-S265A/S284A are more sensitive to doxorubicin

** Numb WT AA DD ** DOX - + - + - + *** *

) 20 Flag 5 15

p53 10 b-actin 5

Cell # (1 x 10 x (1 # Cell 0 p53/b-actin 1.0 5.1 3.8 9.1 1.6 4.1 MCF cells

* Numb WT AA DD * 60 * DOX - + - + - + * **

) **

Flag 4 50 10 × 40 p53 30

Cleaved PARP Number (1 20

b-actin Cell 10

p53/b-actin 1.0 8.5 2.6 11.3 1.3 6.1 0 U2OS cells Cells expressing Numb-S265A/S284A are more sensitive to doxorubicin

- DOX + DOX

WT ** 250 * 200

150 AA 100

50 # of colonies of #

0 WT+DOX AA+DOX DD+DOX DD Tumors carrying Numb-S265A/S284A are more sensitive to chemotherapy

500

450

400 FLAG β-actin 350

300 WT DOX injection AA 250 DD 200

WT+DOX volume (mm3) volume 150 AA+DOX 100 DD+DOX ** Tumor Tumor * 50 * 0 0 5 10 15 20 25 30 35 40 45 50 55 Days post-inoculation

600 *

WT WT+DOX 500 * 400 300

AA AA+DOX 200 Tumor weight(mg) Tumor 100 0 DD DD+DOX Tumors carrying Numb-S265A/S284A are more sensitive toFigure chemotherapy 7 a

WT S265A/S284AAA S265D/S284DDD

20 * 18 16

DOX 14

- 12 10 8 WT+DOX AA+DOX DD+DOX cells(%) tumor Positive 6 4 2

0 + DOX DOX +

Cleaved Caspase 3 IHC Plk1 phosphorylatesWorking model MDC1 at T4

Publications: Shao et al., Oncogene 2018, 37, 810.

Tumors carrying a high level of Plk1 and WT p53 Plk1

P P Dr. Chen Shao S265 S284 Numb p53 Low response Numb p53 Degradation Degradation to chemotherapy

Plk1 BI2536

S265 S284 Numb p53 Enhanced response Numb p53 Stabilization Stabilization to chemotherapy Acknowledgements

Funding: NIH