Identification and Characterisation of Biomarkers of Chemoresistance in Breast Cancer

Athanasios Karapetsas, MSc PhD Senior Research Associate

MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, UK

MRC Protein Phosphorylation and Ubiquitylation Unit Breast Cancer

• The leading type of cancer in women (25% of all cases).

• 1 in 8 women will be diagnosed with breast cancer.

•Risk factors: ionizing radiation, lifestyle, hormone replacement therapy, family history.

• 5-10% of cases due to genetic predisposition (mutations in BRCA1&2 genes inherited from parents).

• Aberrant regulation of signaling pathways in breast tumors. Global cancer statistics 2020, Sung et al 2021

MRC Protein Phosphorylation and Ubiquitylation Unit

Signal Transduction - Kinases

Signal Receptor Y

Cell membrane

Transduction molecules

Cellular response

MRC Protein Phosphorylation and Ubiquitylation Unit Signal Transduction - Kinases

Signal Receptor Y

Cell membrane

Transduction molecules

Cellular response

MRC Protein Phosphorylation and Ubiquitylation Unit Signal Transduction - Kinases

MRC Protein Phosphorylation and Ubiquitylation Unit PI3K Signaling pathway

• Key signaling pathway regulating cell growth, survival, proliferation and metabolism.

• PI3K Class I, II & III

• PI3K Class I  PtdIns (3,4,5)P3

(plasma membrane)

•PI3K Class I  PtdIns (3)P

• Recruitment of adaptor- effector proteins to the PtdIns

MRC Protein Phosphorylation and Ubiquitylation Unit PI3K Signaling and Breast Cancer

• Most tumors harbor mutations that hyperactivate the PI3K pathway.

Gene Alterations Effect on the HR+ HER+ TNBC pathway IGFR Activating Activation of IGF-R 41-48% 18-64% 42% mutations, gene signaling pathway amplification PIK3CA Activating Activation of PI3K 8-26% 23-42% 8-26% mutations, signaling pathway gene amplification PTEN Loss of Activation of PI3K 4-44% 5-22% 8-26% heterozygosity signaling pathway Akt Activating Activation of Akt 2-4% 0-2% 0-1% mutation McKenna, McGarrigle and Pidgeon, 2018

MRC Protein Phosphorylation and Ubiquitylation Unit PI3K pathway inhibitors

McKenna, McGarrigle and Pidgeon, 2018

• Many tumors exhibit inherent or develop acquired resistance to PI3K inhibitors.

• Subset of tumors that are PI3K/PDK1-dependent but AKT independent.

MRC Protein Phosphorylation and Ubiquitylation Unit Growth factors

PI3K

PTEN

PDK1 mTORC2

Akt

TSC1/2

mTORC1

S6K 4EBP1 Cyclin D Grb10

Translation, Ribosome biosynthesis, Autophagy, MRC Protein Phosphorylation and Ubiquitylation Unit Feedback loops Growth factors

PI3K

PTEN • Prolonged inhibition of PI3K and/or Akt results in great pressure to activate a PDK1 mTORC2 mechanism that will substitute for Akt loss activity.

Akt

TSC1/2

mTORC1

S6K 4EBP1 Cyclin D Grb10

Translation, Ribosome biosynthesis, Autophagy, MRC Protein Phosphorylation and Ubiquitylation Unit Feedback loops Growth factors

PI3K

PTEN • Prolonged inhibition of PI3K and/or Akt results in great pressure to activate a PDK1 mTORC2 mechanism that will substitute for Akt loss activity.

SGK Akt

TSC1/2 Up-regulation of the Serum- and Glucocorticoid- regulated mTORC1 Kinases, SGKs

S6K 4EBP1 Cyclin D Grb10

Translation, Ribosome biosynthesis, Autophagy, MRC Protein Phosphorylation and Ubiquitylation Unit Feedback loops Serum - and Glucocorticoid - regulated Kinases, SGKs

PDK1 mTORC2

T308 S473 1 480 •Belong to the AGC superfamily of C-term H Akt PH kinase Ser/Thr kinases. regulatory

T253 S416 • 3 family members: SGK1, SGK2 & 1 431 SGK3. C-term H SGK1 kinase regulatory • High similarity to Akt. Like Akt, they phosphorylate Ser/Thr residues in the T256 S422 1 427 RxRxxS/Tx motif.

SGK2 C-term H kinase regulatory Akt has preference for large T320 S486 hydrophobic residues (Leu, 1 496 Ile, Phe) in the +1 position C-term H from the phosphorylation site. SGK3 PX kinase regulatory

MRC Protein Phosphorylation and Ubiquitylation Unit Breast cancer cell lines carrying mutations in PTEN or PI3K that also express high levels of SGK1, display inherent resistance to PI3K or Akt inhibitors

Cell line MK-2206 AZD5363 GI50 GI50

BT-474 0.2 0.14 CAMA1 0.58 0.04 ZR-75-1 0.69 1.5

Sensitive T47D 0.09 0.14 HCC-1187 0.88 0.61 SUM52PE 0.18 1.1 HCC-1937 >20 >20

MDA-MD-436 9.7 14.9 BT-549 4.5 >20 MDA-MB-157 >20 >20

Resistant HCC-1806 >20 >20 MDA-MB-231 >20 >20 JIMT-1 >20 >20 Sommer et al, 2013 MRC Protein Phosphorylation and Ubiquitylation Unit Prolonged inhibition of PI3K/Akt in breast cancer cells results in up-regulation of SGK3 that substitutes for Akt.

SGK Akt

TSC1/2

mTORC1

S6K 4EBP1 Cyclin D Grb10

MRC Protein Phosphorylation and Ubiquitylation Unit Bago et al, 2016 PDK1-SGK Signaling sustains Akt-independent mTORC1 activation and confers resistance to PI3K inhibition in mouse models

Castel et al, 2016

MRC Protein Phosphorylation and Ubiquitylation Unit Dual treatment with Akt and SGK inhibitors reduces tumour growth in BT-474 xenograft mouse model

Bago et al, 2016 MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3

MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3

•IGF1 stimulation enhances endosomal PtdIns(3)P and localisation of SGK3 to the Plasma membrane PI3K endosomes (Malik et al, 2018). Class I PI3K Class III •Both Class I and Class III PI3Ks (Vps34) mediate IGF-induced activation of SGK3 (Malik et al, 2018).

•SGK3 phosphorylation and its kinase activity is controlled by its Endosome binding to PI(3)P (Bago et al, 2014).

•SGK3 is localized on the endosomes and upon Vps34 inhibition is dispersed in the cytoplasm (Bago et al, 2014).

MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3 Growth factors

•IGF1 stimulation enhances endosomal PtdIns(3)P and localisation of SGK3 to the Plasma membrane PI3K endosomes (Malik et al, 2018). Class I PI3K Class III •Both Class I and Class III PI3Ks (Vps34) mediate IGF-induced activation of SGK3 (Malik et al, 2018).

•SGK3 phosphorylation and its kinase activity is controlled by its Endosome binding to PI(3)P (Bago et al, 2014).

•SGK3 is localized on the endosomes and upon Vps34 inhibition is dispersed in the cytoplasm (Bago et al, 2014).

MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3 Growth factors

•IGF1 stimulation enhances endosomal PtdIns(3)P and localisation of SGK3 to the Plasma membrane PI3K endosomes (Malik et al, 2018). Class I PI3K Class III •Both Class I and Class III PI3Ks (Vps34) mediate IGF-induced activation of SGK3 (Malik et al, 2018).

•SGK3 phosphorylation and its PI(3)P kinase activity is controlled by its PI(3)P Endosome binding to PI(3)P (Bago et al, 2014). PI(3)P PI(3)P •SGK3 is localized on the endosomes and upon Vps34 inhibition is dispersed in the cytoplasm (Bago et al, 2014).

MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3 Growth factors

•IGF1 stimulation enhances endosomal PtdIns(3)P and localisation of SGK3 to the Plasma membrane PI3K endosomes (Malik et al, 2018). Class I PI3K Class III •Both Class I and Class III PI3Ks (Vps34) mediate IGF-induced activation of SGK3 (Malik et al, 2018).

•SGK3 phosphorylation and its PI(3)P kinase activity is controlled by its PI(3)P Endosome binding to PI(3)P (Bago et al, 2014). T320 PX PI(3)P PDK1 P SGK3 PI(3)P •SGK3 is localized on the P mTORC2 endosomes and upon Vps34 S486 inhibition is dispersed in the cytoplasm (Bago et al, 2014).

MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3 Growth factors

•IGF1 stimulation enhances endosomal PtdIns(3)P and localisation of SGK3 to the Plasma membrane PI3K endosomes (Malik et al, 2018). Class I PI3K Class III •Both Class I and Class III PI3Ks (Vps34) mediate IGF-induced activation of SGK3 (Malik et al, 2018).

•SGK3 phosphorylation and its PI(3)P kinase activity is controlled by its PI(3)P Endosome binding to PI(3)P (Bago et al, 2014). T320 PX PI(3)P PDK1 P SGK3 PI(3)P •SGK3 is localized on the P mTORC2 endosomes and upon Vps34 S486 inhibition is dispersed in the cytoplasm (Bago et al, 2014).

P

NDRG1

MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3 Growth factors

•IGF1 stimulation enhances endosomal PtdIns(3)P and localisation of SGK3 to the Plasma membrane PI3K endosomes (Malik et al, 2018). Class I PI3K Class III •Both Class I and Class III PI3Ks (Vps34) mediate IGF-induced activation of SGK3 (Malik et al, 2018).

•SGK3 phosphorylation and its PI(3)P kinase activity is controlled by its PI(3)P Endosome T308 T320 PX PI(3)P binding to PI(3)P (Bago et al, 2014). PH PDK1 P P PI(3)P Akt SGK3 •SGK3 is localized on the P P mTORC2 endosomes and upon Vps34 S473 S486 inhibition is dispersed in the cytoplasm (Bago et al, 2014).

P

P NDRG1 PRAS40 P Most known substrates TSC2 are shared with Akt

MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3 Growth factors

•IGF1 stimulation enhances endosomal PtdIns(3)P and localisation of SGK3 to the Plasma membrane PI3K endosomes (Malik et al, 2018). Class I PI3K Class III •Both Class I and Class III PI3Ks (Vps34) mediate IGF-induced activation of SGK3 (Malik et al, 2018).

•SGK3 phosphorylation and its PI(3)P kinase activity is controlled by its PI(3)P Endosome T308 T320 PX PI(3)P binding to PI(3)P (Bago et al, 2014). PH PDK1 P P PI(3)P Akt SGK3 •SGK3 is localized on the P P mTORC2 endosomes and upon Vps34 S473 S486 inhibition is dispersed in the cytoplasm (Bago et al, 2014).

P

P NDRG1 ? PRAS40 P Most known substrates TSC2 Are there any SGK3- are shared with Akt selective substrates?

MRC Protein Phosphorylation and Ubiquitylation Unit Regulation of SGK3 Growth factors

•IGF1 stimulation enhances endosomal PtdIns(3)P and localisation of SGK3 to the Plasma membrane PI3K endosomes (Malik et al, 2018). Class I PI3K Class III •Both Class I and Class III PI3Ks (Vps34) mediate IGF-induced activation of SGK3 (Malik et al, 2018).

•SGK3 phosphorylation and its PI(3)P kinase activity is controlled by its PI(3)P Endosome T308 T320 PX PI(3)P binding to PI(3)P (Bago et al, 2014). PH PDK1 P P PI(3)P Akt SGK3 •SGK3 is localized on the P P mTORC2 endosomes and upon Vps34 S473 S486 inhibition is dispersed in the cytoplasm (Bago et al, 2014).

P

P NDRG1 ? PRAS40 P Most known substrates TSC2 Are there any SGK3- are shared with Akt selective substrates?

Biomarkers of SGK3 MRC Protein Phosphorylation and Ubiquitylation Unit activity Identification and characterization of biomarkers – Workflow

1. Omics – 2. In vitro validation 3. In vivo validation 4. Development of Identification of of targets of targets reagents and targets/hits assays

MRC Protein Phosphorylation and Ubiquitylation Unit 1. Omics – 2. In vitro validation 3. In vivo validation 4. Development of Identification of of targets of targets reagents and targets/hits assays

1. Identification of targets

MRC Protein Phosphorylation and Ubiquitylation Unit 1. Omics – 2. In vitro validation 3. In vivo validation 4. Development of Identification of of targets of targets reagents and targets/hits assays

1. Identification of targets

Phosphoproteomics

MRC Protein Phosphorylation and Ubiquitylation Unit Quantitative phosphoproteomics using TMT (Tandem mass tag) labelling to identify selective SGK3 substrates.

MRC Protein Phosphorylation and Ubiquitylation Unit Models of study

Genetic Pharmacological

Wild type cells Wild type cells treated with:

vs MK-2206

SGK3 Knock-out cells AKT (gene editing by CRISPR/Cas9)

or

14h

SGK3

MRC Protein Phosphorylation and Ubiquitylation Unit Quantitative phosphoproteomics using TMT (Tandem mass tag) labelling to identify selective SGK3 substrates.

PS1 PS2

MRC Protein Phosphorylation and Ubiquitylation Unit Quantitative phosphoproteomics using TMT (Tandem mass tag) labelling to identify selective SGK3 substrates.

• No significant changes at total proteome level

MRC Protein Phosphorylation and Ubiquitylation Unit Identification of novel SGK3 selective substrates.

MRC Protein Phosphorylation and Ubiquitylation Unit Identification of novel SGK3 selective substrates.

MRC Protein Phosphorylation and Ubiquitylation Unit 1. Omics – 2. In vitro validation 3. In vivo validation 4. Development of Identification of of targets of targets reagents and targets/hits assays

2. In vitro validation

MRC Protein Phosphorylation and Ubiquitylation Unit 1. Omics – 2. In vitro validation 3. In vivo validation 4. Development of Identification of of targets of targets reagents and targets/hits assays

2. In vitro validation

In vitro kinase assays with recombinant proteins and radiactive- labelled ATP

MRC Protein Phosphorylation and Ubiquitylation Unit SGK3 but not Akt phosphorylates STX7, STX12, RFIP4 and WDR44 in vitro

* *

Similar results obtained for: MPST1 ITSN1 ATXN1 PANK4 AFF4 BABAM1

MRC Protein Phosphorylation and Ubiquitylation Unit SGK3 but not Akt phosphorylates STX7, STX12, RFIP4 and WDR44 in vitro

* * * *

Similar results obtained for: Similar results obtained for: MPST1 STX7 (Ser126) ITSN1 ATXN1 RFIP4 (Ser527) PANK4 AFF4 WDR44 (Ser344) BABAM1

MRC Protein Phosphorylation and Ubiquitylation Unit 1. Omics – 2. In vitro validation 3. In vivo validation 4. Development of Identification of of targets of targets reagents and targets/hits assays

3. In vivo validation

MRC Protein Phosphorylation and Ubiquitylation Unit The Syntaxins

S128 P Syntaxins (STXs): SNARE proteins (N- N-terminal SNARE TM ethylmaleimide-sensitive factor STX7 regulatory attachment protein receptor) that S139 mediate cellular trafficking, membrane P fusion and vesicle release. N-terminal SNARE TM STX12 regulatory

STX7: Part of the SNARE complex comprising syntaxin 8, Vti1b and VAMP8. Involved in the fusion of late endosomes with lysosomes.

STX12: Localized in early and recycling endosomes and the plasma membrane. Interacts with syntaxin 6, Vti1a and VAMP4. Responsible for the homotypic fusion of early endosomes

MRC Protein Phosphorylation and Ubiquitylation Unit Phos-tag gel electrophoresis

Sample 1 Sample 2 Sample 3 Sample 4

P P P P Protein X Protein X Protein X Protein X Protein X

Normal SDS gel electrophoresis

MRC Protein Phosphorylation and Ubiquitylation Unit Phos-tag gel electrophoresis

Sample 1 Sample 2 Sample 3 Sample 4

P P P P Dinuclear metal complex Protein X Protein X Protein X Protein X Protein X that bind s phosphate groups

Normal SDS gel electrophoresis

MRC Protein Phosphorylation and Ubiquitylation Unit Phos-tag gel electrophoresis

Sample 1 Sample 2 Sample 3 Sample 4 Sample 1 Sample 2 Sample 3 Sample 4

P P Protein X

P P P Protein X Protein X Protein X

P P P P Protein X Protein X Protein X Protein X Protein X Protein X Protein X Protein X

Normal SDS gel electrophoresis Phos-tag SDS gel electrophoresis

• No need for phospho-specific antibodies

• Determination of the stoichiometry of the reaction

MRC Protein Phosphorylation and Ubiquitylation Unit SGK3-dependent phosphorylation of endogenous STX7 and STX12 in vivo.

phosphorylated

non-phosphorylated

MRC Protein Phosphorylation and Ubiquitylation Unit SGK3-dependent phosphorylation of endogenous STX7 and STX12 in vivo.

phosphorylated

non-phosphorylated

MRC Protein Phosphorylation and Ubiquitylation Unit Inhibition of upstream activators of SGK3 blocks phosphorylation of STX7 and STX12 in cells.

Similar results were obtained for STX12 MRC Protein Phosphorylation and Ubiquitylation Unit IGF-dependent phosphorylation of STX7 at Ser126 and of STX12 at Ser139 by SGK3.

MRC Protein Phosphorylation and Ubiquitylation Unit 1. Omics – 2. In vitro validation 3. In vivo validation 4. Development of Identification of of targets of targets reagents and targets/hits assays

4. Development of reagents and assays

MRC Protein Phosphorylation and Ubiquitylation Unit 1. Omics – 2. In vitro validation 3. In vivo validation 4. Development of Identification of of targets of targets reagents and targets/hits assays

4. Development of reagents and assays

• Phos tag gel analysis

•Development of phospho-specific antibody

MRC Protein Phosphorylation and Ubiquitylation Unit Generation of phospho-specific antibody against phospho-STX12 Ser139.

A C

B

MRC Protein Phosphorylation and Ubiquitylation Unit What is the biological role of STX12 phosphorylation by SGK3?

MRC Protein Phosphorylation and Ubiquitylation Unit What is the biological role of STX12 phosphorylation by SGK3?

• Phosphorylation-dependent interactions?

• Subcellular localisation?

MRC Protein Phosphorylation and Ubiquitylation Unit Phosphorylation of STX12 at Ser139 by SGK3 enhances the association with the SNARE complex.

MRC Protein Phosphorylation and Ubiquitylation Unit Analysis of STX12 localisation upon IGF stimulation

GFP-STX12 WT GFP-STX12 S139A

-IGF1 +IGF1 +IGF1 +14H -IGF1 +IGF1 +IGF1 +14H

DA

/

PI

GFP

DAPI

/ /

Rab5

Merge

10μM Rab5: Early endosomes

MRC Protein Phosphorylation and Ubiquitylation Unit Phosphorylation of STX12 at Ser 139 by SGK3 promotes its localisation to the plasma membrane

GFP-STX12 WT

-IGF1 +IGF1 +IGF1 +14H

DA

/ PI

4000000

GFP

3000000 DAPI

/ 2000000

STX12 STX12

- Na Na

GFP 1000000

ATPase

Plasma membrane Plasma (Sum intensity/nuclei) (Sum 0 -IGF1 +IGF1 +IGF1

+14H Merge

MRC Protein Phosphorylation and Ubiquitylation Unit Quantification by Alan Prescott Conclusions-Discussion

• By employing TMT phosphoproteomics we identified the first in vivo SGK3 selective substrates, namely STX7 and STX12. • Phosphorylation of STX12 by SGK3 enhances its interaction with the SNARE partners and its localisation to the plasma membrane. • Monitoring phosphorylation of STX7/STX12 or other substrates could be used as biomarker of SGK3 activity.

Development of an essay for the identification of AKT-inhibitor resistant but SGK3-dependent tumors

MRC Protein Phosphorylation and Ubiquitylation Unit Thank you

MRC Protein Phosphorylation and Ubiquitylation Unit