The Role of Nucleus Mechanical Properties in

Senescence-Induced Breast Cancer Cells

2020.06.29
SAMA

Applied Mechanobiology Group

 Senescence

Applied Mechanobiology Group

 Pro-tumorigenic Effects of Senescence-
associated Secretory Phenotype (SASP)

Wang B, Kohli J, Demaria M. Senescent Cells in Cancer Therapy: Friends or Foes? [published online ahead of print, 2020]. Trends Cancer

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 Epithelial Mesenchymal Transition (EMT)

Markers associated Markers associated with

with the epithelial the mesenchymal cell
cell state state

E-cadherin Fibronectin
Claudin Vimentin
Occludin N-cadherin
Cytokeratin Vitronectin
α6β4 Integrin β1 and β3 Integrins
ZO1 α5 Integrin
MMPs

Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019
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 EMT-Inducing Core Transcription Factors
EMT-TFs directly binding to E-cadherin promoter (Through E-box) EMT-TFs indirectly regulate E-cadherin expression

• ZEB1/ZEB2 (Zinc-finger E-box-binding homeobox factors) TWIST1/TWIST2 (Basic helix-loop-helix factors)

• SNAIL (SNAI1)
• SLUG (SNAI2)

Wnt, TGFβ and Notch Signaling Pathways

Mitogenic Growth Factors
Cytokines and Chemokines in Tumor Microenvironment

Regulate

EMT-inducing Transcription Factors ZEB1/ZEB2 Snail/Slug TWIST

EMT
Epithelial State Mesenchymal State

Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019
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 Senescence-Associated Interleukin-6 and EMT Induction

Interleukin-6

Janus Kinase (JAK1/2)

Signal Transducer and Activator of Transcription (STAT3)

Nuclear localization of phosphorylated STAT3

Snail and TWIST1 activation

EMT Induction

Molecular Pathways: Linking Tumor Microenvironment to Epithelial–Mesenchymal Transition in Metastasis. Hae-Yun Jung, Laurent Fattet and Jing Yang.

Clin Cancer Res March 1 2015
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 Senescence !!! Tumorigenesis Promoting or Suppressing ?

Cancer Therapy Chemoresistance

ion
lut
So

Inducing Senescence

Growth Arrest Senescence-associated secretory

phenotype (SASP)

Tumor Suppressor Tumor promotion

EMT Induction

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 Importance of Senescent Cells in
Cancer Therapy

Tumor Cells Chemoresistance

Cancer Therapy n
lu tio
So

Senescent Tumor Cells Senolytic Therapy

Immune Clearance Promotion of Tumor Growth

EMT Induction

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 Research Methods

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 Senescence Induction

Inducing Senescence

DNA Damage

Check

Senescence Biomarkers (SAβGAL, p21, p16)

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 Senescence Induction
1. Induction of Senescence in Breast Cancer Cell lines :

• Using Senescence-conditioned media treatment

MCF-7
Media enriched with
Senescence-associated IL-6 and IL-6 1

• Drug treatment: doxorubicin2

SAβGAL

2. Examine SAβGAL expression Level:

β -Galactosidase staining:
SPiDER-βGal 3

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 Nucleus Mechanical Properties in Breast Cancer cells
and Senescent Breast Cancer Cells

1. Examine the expression level of major architectural proteins of the nucleus such as
lamin A, lamin B1 in senescent breast cancer cells

2. DNA repair level in confined spaces in senescent breast cancer cells compared to
breast cancer cells

• Using microchannel assays by conducting constrictions vary in shape and length

2. Investigating the probability of senescence induction in cancer cells passing

through confined spaces

• Using microchannel assays by conducting constrictions vary in shape and length to check
the occurrence of senescence via DNA damages caused buy nuclear envelope rupture
when cells migrate through confined spaces

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 SASP Factors Effect on Surrounding Cells

1. Co-culture
• Using transwell culture plates 1

2. EMT induction examination through Cell migration and invasion assays

1
Cell Migration: transwell insert
Invasion: matrigel-coated transwell insert

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 Long-term Objective:
To characterize the role of nucleus mechanical properties in
senescence-induced breast cancer cells to find probable targets for
senolytic therapy

Applied Mechanobiology Group

 THANK YOU

Applied Mechanobiology Group

 Markers of Senescence

- Reactivation of SA-β-gal or SABG: senescence-associated β-galactosidase

- High expression level of

p16INK4a (CDKN2A): cyclin-dependent kinase (CDK) inhibitor CDK4 and CDK6 kinases G1 to S-phase progression

- Induction of SASP factors (Senescence-associated secretory phenotype) : pro-inflammatory cytokines and chemokines
secreted by senescent cells effecting surrounding cells

- High expression level of H3K9me3

- Lack of cell-cycle associated Ki67 protein

- Lack of DNA replication

Lee, S., Schmitt, C.A. The dynamic nature of senescence in cancer. Nat Cell Biol. (2019)
Liu JY, Souroullas GP, Diekman BO, et al. Cells exhibiting strong p16INK4a promoter activation in vivo display features of senescence. PNAS 2019
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 Chromatin Remodeling in Senescence

P16 (through ERK/ETS1/2 )

Senescence
1) DNA damage G1-phase cell-cycle inhibitors
inducing factors
P21 (Through p53))
recruit
Rb-E2F complex H3K9 methyl transferase Forming heterochromatin near Inhibits S1 phase
the promoter of EF2 genes

Senescence
2) inducing factors ATM/ATR activation Unresolved DSBs P53 and Rb (retinoblastoma) pathway activation

initiate recruit HIRA and ASF1A establish SAHF (Senescence-associated

PML (promyelocytic leukemia)
chromatin remodelers heterochromatin foci)

Lee, S., Schmitt, C.A. The dynamic nature of senescence in cancer. Nat Cell Biol. (2019)
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Muñoz-Espín, D., Serrano, M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol 15, 482–496 (2014)

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 EMT signaling Pathways

EMT signaling pathways include:

• Canonical TGFβ pathway

• Non-canonical TGFβ pathway
• Canonical WNT signaling
• Non-canonical WNT signaling
• NOTCH signaling
• Mitogenic growth factors

Cellsignal.com

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 TGFβ Pathway in EMT
Canonical (SMAD-dependant) TGFβ/BMP signaling Non-Canonical TGFβ signaling
TGFβ BMP
+ + RTK (Receptor tyrosine kinase)
TGFβR1/2 TGFβR1/2

heterotetrameric ligand-receptor complex

PI3K Ras/Raf
Recruit and phosphorylate

SMAD2,SMAD3 SMAD1, SMAD5, SMAD8/9

Bind to
GSK-3β MEK
SMAD4
(Common mediator coSMAD)
Β-catenin phosphorylation ERK

Nuclear Translocation

Activation of EMT TFs

Activation of TFs (SNAIL,SLUG)

Cervello M, Augello G, Cusimano A, et al. Pivotal roles of glycogen synthase-3 in hepatocellular carcinoma. Adv Biol Regul. 2017 Cellsignal.com
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 Mitogenic Growth Factors and EMT Induction
EGF: epidermal growth factor  activating EMT through:

Nuclear localization of phosphorylated SMAD2/3

Autocrine positive feedback loop EGF Nuclear localization of SNAIL

by contaminant increase with IL-6

EGFR JAK2

MEK-ERK STAT3

Decreased TWIST activation

expression of E-
cadherin

EGFR pathway activation  -fibroblast-like morphology

-expression of vimentin and N-cadherin

Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019
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 Mitogenic Growth Factors and EMT Induction
FGF: Fibroblast growth factor  activating EMT HGF: Hepatocyte growth factor 
through: -activating EMT
-enhancing migratory and invasive ability of carcinoma cells
N-cadherin FGF2 FGF1
+ + HGF HGF
+
FGFR FGFR +
FGFR
EMT tyrosine kinase receptor
Phosphorylation of E47
PI3K/Akt MAPK
MEK-ERK
SNAIL
PAK5 (p21-activated kinase 5)
Promote Cancer stem-like Brachyury (T box
metastasis cells formation transcription factor)
Activation of EMT TFs
E-cadherin
EMT

FGFR1 Decreased expression of

SNAIL and fibronectin

PDGF: Platelet-derived growth factor  activating EMT

Mrozik KM, Blaschuk OW, Cheong CM, Zannettino ACW, Vandyke K. N-cadherin in cancer metastasis, its emerging role in haematological malignancies
and potential as a therapeutic target in cancer. BMC Cancer. 2018
Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019
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 Similar molecular functions
E-Cadherin N-Cadherin

Forming stable adherent junctions Weak adherent junctions

Strong cell-cell contact Enhances cell migration

During EMT: Downregulation Upregulation

Loh CY, Chai JY, Tang TF, et al. The E-Cadherin and N-Cadherin Switch in Epithelial-to-Mesenchymal Transition: Signaling, Therapeutic Implications, and
Challenges. Cells. 2019

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 DNA DAMAGE

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 Ataxia Telangiectasia Mutated (ATM) and DDR

DNA double strand breaks

ATM Activation

DNA damage checkpoints activation

protein post-translational modifications (PTMs)

(phosphorylation of key proteins by ATM)

signal transduction activates downstream effectors

(P53, H2AX, …)

cell survival apoptosis

Shiloh, Y., Ziv, Y. The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol. 2013

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 ATM and ATR

ATR: ATM and RAD3-related

DSB  phosphorylation of H2AX on serine 139(called γH2AX) RPA: Replication protein A

MRN: sensory complex which detects DSBs 9-1-1 complex: Rad9-Hus1-Rad1

BRCA1: Breast Cancer Susceptibility Gene 1

Nilsson K, Wu C, Schwartz S. Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation. Int J Mol Sci. 2018

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 Tissue Dependent Role of EMT-TFs in DDR

Radiotherapy  induce DNA Damage in cancer cells  leads to induce cancer cell death

EMT-TFs DNA Damage Radioresistancy of cancer cells

ZEB1 ATM-mediated DNA Damage repair1 (Lung cancer)

ATM expression2

ZEB2 Similar effects as ZEB1 in bladder cancer

Snail/Slug ATM-mediated DNA Damage repair 3 (Breast cancer)

TWIST1 DNA Damage response

Nieto MA, Huang RY, Jackson RA, Thiery JP. EMT: 2016. Cell. 2016
Stemmler, M.P., Eccles, R.L., Brabletz, S. et al. Non-redundant functions of EMT transcription factors. Nat Cell Biol 

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 DSBs Repair Pathways

Cells choose different repair pathways according to the requirement for substantial DNA end resection
DNA End Resection: generation of 3’ single stranded DNA (ssDNA) by nucleolytic degradation of broken ends
 facilitates end joining

NHEJ: non-homologous end joining SSA: single strand annealing

a-EJ: alternative end joining HR: homologous recombination

Liu T, Huang J. DNA End Resection: Facts and Mechanisms. Genomics Proteomics Bioinformatics. 2016
Chang, H., Pannunzio, N., Adachi, N. et al. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol (2017)
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 Factors involved in DSBs Repair Pathways

53BP1 (p53‑binding protein 1): positive regulator of NHEJ (remodeling chromatin)

DNA-PKcs : DNA-dependent protein kinase catalytic subunit NHEJ
Artemis: a nuclease and is thought to be required only for repair of DSBs with damaged ends

CtIP: carboxy‑terminal binding protein interacting protein HR, a-EJ, SSA

MRN: MRE11–RAD50–NBS1 (Nijmegen breakage syndrome protein 1) complex

PARP1: Poly(ADP-ribose)polymerase 1
Pol θ : DNA polymerase θ
a-EJ

BLM: Bloom syndrome RecQ-like helicase

EXO1: exonuclease 1 HR, SSA
RPA: replication protein A

RAD52: annealing of long regions of homology

(XPF)-ERCC1:  xeroderma pigmentosum group F complex
SSA

RAD51: strand exchange

RAD54, BRCA1, BRCA2 HR

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 IL-6 and IL-8 in Breast Cancer Cells

MCF-7 treatment with IL6 and IL8 induced morphological and phenotypic changes associated with an EMT process

Upregulation of EMT-TFs in IL-6 and

IL-8 treated cells

MCF-7 cells treated with IL_6 and IL-8  increased

migration and invasion capacity (wound-healing assay)

Ortiz-Montero, P., Londoño-Vallejo, A. & Vernot, J. Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced
senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line. Cell Commun Signal 15, 17 (2017)

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