Basics of metastasis and emt

1. Metastatic cascade and
Epithelial Mesenchymal Transition
Presenter – Dr Shruti Dogra
Moderator – Dr P. Malhotra

2. What is cancer metastasis?
• Cancer is defined as
•A population of cells that have lost their normal
controls of growth and differentiation
•Proliferating without check.
• Metastasis
•The process by which a tumor cell leaves the primary
tumor Travels to a distant site via the
circulatory system Establishes a secondary
tumor.

3. Why do we need to know the metastatic
cascade?
• It is estimated that metastasis is responsible for
about 90% of cancer deaths.
• About 1,500 people continue to die each day from
cancer due to the failure in managing the disease once it
disseminates through the body.
• Metastasis remains a final frontier in the search for a
cure for cancer.

4. • Every tumor’s:
- potential to metastasise is different
- metastatic sites are different
- same histological grade of tumor may show different
metastatic behaviour
• Study metastasis in detail to outline strategies to curb it.

5. Metastasic
Cascade
• Invasion and passage
through
basement membrane
• Migration through
extracellular matrix
• Migration into vessels
(intravasation)

6. • Adhesion to vascular
endothelium (usually in lung
or liver)
• Extravasation and migration
into tissue
• Establishment and growth
at new site

7. STEP I - Invasion
1. “Loosening up” of tumor cell-tumor cell interactions.
2. Degradation of ECM
3. Attachment to novel ECM components
4. Migration and invasion of tumor cells

8. 1. “Loosening up” of tumor cells:
• Alteration in intercellular adhesion molecules

9. Role of E Cadherin in invasion
-Cell surface protein – Intercellular adhesiveness
-β catenin protein
binds to the
cytoplasmic tail of
E Cadherin

10. Wound/Injury
Loss of cell-cell contact
Diruption of E Cadherin – β catenin interaction
Increased translocation of β catenin to the nucleus
Promotes proliferation and repair

11. • Reestablishment of E Cadherin - β catenin as wound
heals reduces the proliferative signal.
• These cells are said to be “contact inhibited”
Mutation/ Loss of E Cadherin
Loss of contact
inhibition
Easy disaggregation
of cells
Malignant phenotype – Invade & Metastasise

12. 2. Degradation of ECM:
• Proteolytic enzymes - Matrix Metalloproteinases (MMP’s)
- Cathepsins
- Plasmin-Plasminogen activator

13. MMP’s
• Multigene family of zinc dependent extracellular matrix
(ECM) remodeling endopeptidases
• Produced as inactive precursors (Zymogens)
• Activated by cleavage of a propeptide
• Rapidly inhibited by specific tissue inhibitors of
metalloproteinase (TIMP’s) – Mesenchymal cells

14. • MMP 1, 2 & 3
•Interstitial collagenase
•Cleave fibrillar collagen
• MMP 2 & 9
•Gelatinase
•Degrade amorphous collagen and fibronectin
• MMP 3, 10 & 11
•Stromelysins
•Degrade variety of ECM components ( proteoglycans,
laminin, fibronectin )

15. Other functions of MMPs in cancer
• MMPs Affect Growth Signals – like TGF β, EGFR
• MMPs Regulate Apoptosis – cleavege of ligands (Fas)
that induce apoptosis.
• Tumor angiogenesis – MMP 9 has distinct role by
regulating the bioavailability of vascular derived
endothelial growth factors (VEGF).

16. Cathepsins
• Diverse group
• Serine proteases
• Cysteine proteases
• Aspartic proteases
• Synthesized as inactive precursors.
• Activated at acidic pH in the lysosomes.
• Contribution to invasion is well documented.
• Exact mechanism still not clear.

17. Cathepsin Cancer
A Malignant melanoma
B Breast, Lung, Gastric, Pancreatic, Bladder
D Thyroid, Renal, Ovarian, SCC
E Panreatic ductal carcinoma, Gastric
F Cervical carcinoma
G Breast
H Breast, Colorectal, Prostate
K Gastric, SCC, BCC
L Breast, Lung, Gastric
S Astrocytoma, Gastric, Hepatocellular
X Prostate, Gastric, Malignant melanoma
Z Melanomas, Gastric, Hepatocellular

18. Plasmin-Plasminogen activator
• Plasmin - ability to degrade several matrix components
like gelatin, fibronectin, laminin.
- activates MMP’s by propeptide cleavage.
- synthesized in its inactive form plasminogen
- conversion needs plasminogen activator

19. • Plasminogen activator – Two types
1. Urokinase (uPA)
2. Tissue (tPA)
-Plasminogen activator inhibitor (PAI) 1&2
- Involved in regulation of plasmin and pro MMP’s

20. Plasminogen
Plasmin
Pro MMP Active MMP
• Dissolution of basement membrane
& interstitial matrix
• Cell signaling and migration
• Angiogenesis
uPA/tPA

21. • Matrix is modified to promote invasion.
• Cleavage of basemement membrane proteins
- Collagen IV and laminin by MMP 2 or 9
- Generates novel sites
- Bind to receptors on tumor cell and stimulate
migration.
3. Attachment to novel ECM components:

22. 4. Migration and Invasion of Tumor cells:

23. • It is a multistep process.
• Cells must attach to the matrix at leading edge
• Detach from the trailing edge
• Contract the cytoskeleton to rachet forward.
• This is accomplished by epithelial to mesenchymal
transition (EMT).

24. • An orchestrated series of events
• Cell-cell and cell-extracellular matrix (ECM)
interactions are altered.
• Releasing epithelial cells from the surrounding tissue.
• The cytoskeleton is reorganized to allow movement in 3
dimensions in the ECM.
• New transcriptional program is induced to maintain the
mesenchymal phenotype.
Epithelial Mesenchymal Transition

25. • At completion of EMT:
- degradation of underlying basement membrane
- formation of a mesenchymal cell
- can migrate away from the epithelial layer in
which it originated.

27. STEP II - Vascular dissemination of
tumor cells
• In circulation
- Tend to form clumps
- homotypic adhesion among tumor cells
- heterotypic adhesion with blood cells esp. platelets
• Platelet tumor aggregate may enhance tumor cell
survival and implantability.

28. • In circulation tumor cells are vulnerable to destruction
- mechanical shear stress
- apoptosis stimulated by loss of adhesion to basement
membrane (Anoikis)
- immune defense mechanisms

29. Mechanisms by which tumor cells escape immune
recognition
• Low immunogenecity:
- No peptide – No MHC ligand
- No adhesion molecules
- No co stimulatory molecules

30. • Tumor treated as self antigen:
- Tumor antigens taken up and
presented by APC’s are tolerated
by T- cells.

31. • Antigenic modulation:
- Antibody against tumor
cell surface antigens induce
endocytosis and degardation
of antigen.
- Immune selection of antigen
loss variants.

32. • Tumor induced immune suppression:
- Factors (TGF-β) secreted by tumor
cells directly inhibit T-cells.
- Expression of lymphocyte
death receptor ligands
(FasL, TRAIL) by tumor cells.

33. • Tumor induced privilege site:
- Factors secreted by tumor cells create
a physical barrier to the immune system.
- Recruitment and activation of
regulatory T-cells (Tregs – CD4+ CD25+)

34. STEP III - Homing of tumor cells
• Arrest and extravasation of tumor emboli at distant site
- adhesion to the endothelium
- egress through basement membrane
• Site at which circulating tumor cells leave the capillaries
to form secondary deposits
- anatomic location of primary tumor
- vascular drainage of primary tumor
- tropism of particular tumor to specific tissues

35. Preferential metastatic sites
Primary Tumor Common distant sites
Breast adenocarcinoma Bone, Brain, Lung, Adrenal
Prostate adenocarcinoma Bone
Lung small cell carcinoma Bone, Brain, Liver
Skin cutaneous melnoma Brain, Liver, GIT
Thyroid adenocarcinoma Bone
Renal cell carcinoma Bone, Liver, Thyroid
Bladder carcinoma Brain

36. Reason for organ selectivity
• Mechanistic theory: determined by the pattern of
blood flow.
•“Seed and soil” theory: the provision of a fertile
environment in which
compatible tumor cells could
grow.

37. Determining factors for organ tropism
• Compatible adhesion sites on endothelial luminal
surface of target organ.
• Selective chemokine secretion by target tissue for
metastasis.
- Breast carcinoma cells express receptors for chemokine
CXC4 and CCR7.
• Appropriate environment.
- Although well vascularised skeletal muscle and spleen
are rare sites for metastasis.

38. STEP IV - Establishment at distant site
• Accomplished by mesenchymal to epithelial transition
• Colonisation of tumor cells Micrometastasis
• Proliferation and angiogenesis Macroscopic
metastasis

39. Molecular Genetics of Metastasis
development
1. Clonal evolution model:
• Mutations accumulate in genetically unstable cancer
cells, leading to heterogenous population.
• Tumor cell subclones develop gene expression
permissive for EMT and hence to metastasis.

40. 2. Metastasis signature:
• Gene expression studies in breast carcinoma with
increased risk of metastasis found a phenotype which
signified EMT. Genotype ~ Metastasis signature
• These cells with “metastatic signature” have
predilection for metastasis during early stages of
carcinogenesis.

41. 3. Metastatic signature plus an additional mutation are
needed for metastasis to occur.

42. 4. Microenvironment characteristics:
• Stromal response and angiogenesis along with intrinsic
properties of cancer cells promote metastasis.

44. Epithelial
Mesenchymal
Transition

45. Why Epithelial Mesenchymal Transition ?
• EMT is the primary step in the process of metastasis
• Stopping this transition can curb metastasis
-tumors can be resected surgically
- amenable treatment

46. • Has been extensively investigated in past decade
- facilitate development of early detection strategies
- improve therapeutic targeting of malignant tumors

47. Cell Types

48. Characteristics of Epithelial cells:
• Tightly packed cells usually arranged in layers.
• Regularly spaced cell junctions and adhesions between
neighboring cells.
• Tight adhesion between cells resulting in inhibition of
movement away from the monolayer.
• Epithelial cell is polarised i.e. different ends of
cell do different things.

49. • Characterised by specialised membrane
domains:
1- Basal domain - interacts with basement
membrane (BM).
2- Apical domain - depends on the functional
needs of cell.
3- Lateral domain - form adherence and tight
junctions.

50. • A characteristic example is the enterocyte :
- Apical domain: Brush border essential for
resorptive activity.

52. • Apical compartment protein complexes:
•Partitioning defective (PAR) complex
(PAR6, PAR3, atypical protein kinase C (aPKC))
• Crumbs (CRB) complex
• Protein associated with Lin-7 1 (PALS1)
• PALS1 associated tight junction protein (PATJ)

54. -Basal domain: Laminin 5 mediates adhesion to BM
through interaction with integrins &
provides signalling cues from ECM.

56. -Lateral domain: E-Cadherin - catenin complex
( Adherence junctions)
Claudin - Occludin (Tight junctions)

58. Lateral domain of epithelial cells

59. • Basolateral compartment protein complexes:
• Scribble (SCRIB) complex
• Disc large (DLG) complex
• Lethal giant larvae (LGL) complex

61. Characteristics of Mesenchymal cells:
• They lack a regimented structure.
• Very few intracellular adhesions.
• Weak adhesions allow for ease of mobility.
• Front rear cytoplasmic polarity.
• No specific membrane domains.

62. • Cells are potentially mobile
- cytoskeleton composed of vimentin
- myofibroblasts of smooth muscle actin.
• Their secretory activity is targeted towards
production of extracellular matrix components.

63. EPITHELIAL CELL MESENCHYMAL CELL
Arranged in a layers Lack a regimented structure
Many cell junctions and adhesions Very few cell junctions and adhesions
Stationary Ability to migrate
Apical basal polarity Front rear polarity

64. • First observed and defined by Elizabeth Hay in late
1960’s using a model of chicken primitive streak in
embryogenesis.
- Epithelial Mesenchymal Transformation
• Process is reversible with unstable intermediate
EMT Metastable MET
- Hence the term “transition”
Epithelial to Mesenchymal Transition

65. Transitions

66. • Types of EMT
• Three types of EMT:
- Type 1
-Type 2
- Type 3

67. Types of EMT
• Type 1 – EMT during implantation,
embryogenesis and organ development

68. • Type 2 – EMT associated with tissue
regeneration and organ fibrosis

69. • Type 3 – EMT associated with cancer
progression and metastasis

71. Phenotypic modifications associated
with EMT
• Invitro morphology and function
- Stellate or spindle shape
- Resistance to anoikis
- Increased migration
- Invasion into collagen matrix

72. • Down – regulation of epithelilal proteins:
- E Cadherin
- Cytokeratin
- Occludin
- Claudin
Epithelial to Mesenchymal transition

73. • Up – regulation of Mesenchymal proteins:
- N Cadherin
- Vimentin
- Fibronectin
- α Smooth muscle actin
- MMP’s (2, 3, 9)
- Integrin αvβ6
Epithelial to Mesenchymal transition

74. • Transcription factors:
- ZEB
- Smad (2, 3)
- Snai 1
- Snai 2
- Twist
Epithelial to Mesenchymal transition

76. Molecular events comprising EMT
• Deconstruction of cell junctions
• Dissolution of tight junctions
- decreased claudin & occludin expression
- diffusion of zona occludens 1 (ZO1 aka TJP1)
• Destabilization of adherence junctions
- Cleavage of E Cadherin
- Increased nuclear translocation of β catenin

77. • Decreased expression of E Cadherin
- represssion of polarity complex proteins
- loss of apical basal polarity
• Apical basal polarity - organised by polarity complexes
- integrated with cell junctions
• Loss of apical basal polarity

78. • Cytoskeletal changes and motility
• Reorganisation of cortical actin cytoskeleton to enable
cell elongation and directional motility
•Formation of actin rich membrane projections
- Lamellipodia: sheet like
- Filopodia: spike like
- Invadopodia: proteolytic

79. • Actin stress fibre formation – increased cell contractility
• Actin dynamics are regulated by RHO GTPases
- RHO A : Actin stress fibre formation
- RAC1 & CDC42 : lamellopodia & filopodia

80. • To achieve directional polarity:
- PAR, PATJ, Crumbs & Scribble complexes relocalize
to leading edge of cell
- RAC1 & CDC42 activate actin polymerization and
membrane protrusion formation
- RHO A localizes at the rear end & promotes
disassembly of adhesion complexes & cell retraction
• Induces cell shape changes and front rear polarity
essential for migration

83. Transcription factors in EMT
• SNAI family
• TWIST
• ZEB

84. SNAI family of transcription factors
• Three vertebrate SNAI factors
- SNAI 1 aka SNAIL
- SNAI 2 aka SLUG
- SNAI 3 aka SMUC
• Repress epithelial genes by binding to E- box DNA
sequences through carboxy terminal zinc finger
domains.
• Central role in EMT

85. Twist transcripton factor
• Part of basic helix-loop helix (bHLH) transcription
factors.
• Implicated in cell lineage determination and
differentiation.
• Role in Breast carcinoma

86. ZEB transcription factors
• Two vertebrate ZEB factors
- ZEB1 aka TCF8
- ZEB2
• Bind regulatory gene sequences at E boxes
• Repress or activate transcription
• Role Lung carcinoma

87. Signalling pathways in EMT
RECEPTOR LIGAND SIGNALLING
MOLECULES
INTERMEDIATE
SIGNALLING
END POINT
EFFECT
TGF- β
receptor
TGF- β Rho A
Smad family
ZEB Migration
Receptor
Tyrosine
Kinae
FGF
HGF
EGF
Sarc
Ras, MAPK
SNAI2 -Cytoskeleton
activation
-Migration
-Focal adhesion -
rearrangement
Integrins Collagens
Fibronectin
FAK, Paxillin,
Rac
SNAI2 Increased
migration
Frizzled Wnt APC, axin, GSK3β,
β-catenin
SNAI1 E Cadherin down
regulation
Reduced cell
adhesion

89. • Complex interactions between the signalling pathways
during EMT
- gravitate towards downregulation of E Cadherin
- disassembly of junctional complexes.
• Loss of E cadherin expression is associated with
nuclear expression of β catenin via the Wnt
signaling pathway.
• This is associated with the acquisition of stem cell
properties by the cancer cell.

90. Role of E cadherin in EMT
Downregulation of E Cadherin
Disassembly of junctional complexes
Liberates β catenin from
E Cadherin- Catenin
complex
Activation of Wnt
pathway through
LEF/TCF4.
Signals Integrins

91. Activates Rap1 ( GTPase protein)
Activates Integrin
linked kinase (ILK)
Downregulates E Cadherin
& TGF β signaling
Activates
RhoA &
Rac1
Actin
remodelling
Altered
Focal adhesion
kinase (FAK)
Integrins
Resistance
to Anoikis

93. SNAI1 & SNAI2 are the central
regulators
Interaction of FGF, EGF or HGF with their
respective RTK’s
Activates members of GTPase family
(Ras, Rho,Rac)
Activation of of Wnt pathway

94. GSK3β phosphorylates SNAI1
Downregulates E Cadherin expression

95. SNAI1 and SNAI2
Modify pattern of genes for remodelling
cytoskeleton
Upregulation
of Vimentin
expression
Modification
of Integrin
expressionActivation
of contractile
apparatus

96. Epithelial plasticity is bi-directional
• The phenotypic plasticity afforded by an EMT is
revealed by the occurrence of a reverse process –
Mesenchymal epithelial transition.

97. • Disseminated cancer cells undergo MET at the site
of metastasis.
• Explained as EMT derived migratory cancer cells
typically establish secondary colonies at distant sites
that histopathologically resemble the primary tumor
from which they arose.

99. Practical aspects of EMT
• The recent past has seen extensive research on the
different elements of EMT.

104. • The results of these research have lead to the use of
Epithelial Mesenchymal Transition as a powerful tool in:
-Surgical pathology for highlighting tumor invasiveness
- Prognostication of various tumors
- Prediction of therapy resistance
- Potential target for antineoplastic therapies

105. EMT In Surgical Pathology
• Immunohistochemical Markers
• Loss of epithelial markers
- E Cadherin : Colon, Breast, Lung, Ovary,
Esophagus, Prostate, Cervix
- Claudin : Ovary
- Occludin : Esophagus, Breast
Loss of E Cadherin in Breast CA

106. • Loss of E-cadherin expression is associated with
• The switch from noninvasive adenoma to invasive
carcinoma in a transgenic mouse model of
pancreatic β-cell carcinogenesis.

107. • Expression of mesenchymal markers
- N Cadherin : Ovary, Prostate
- Vimentin : Breast, Esophagus, Cervix
- β catenin (Nuclear) : Breast, Cervix
Vimentin in Breast CA

108. • EMT signalling molecules
- SNAI 1 : Breast, Cervix, Ovary
- SNAI 2 : Breast, Ovary
- TWIST : Breast, Stomach
- ZEB 1 : Colon, Breast, Ovary
SNAI 1 in Breast CA

109. • Immunofluoresence markers
-E Cadherin
- Vimentin
- HGF
- TGF β
- NOTCH 1
- Wnt-3a
- BMP 7
Loss of E Cadherin in Breast CA

110. • The single-cell infiltration pattern
• Observed in some lobular carcinomas
• Linked to protein truncation mutations in the CDH1
gene encoding for E-cadherin.
• Tumor cell budding
• Morphologic hallmark of invasive tumor
phenotype and tumor aggressiveness in colorectal
cancer tissue specimens
• Loss of membraneous β-catenin
Histomorphological phenotype associated with EMT

111. EMT and patient prognosis
• The metastatic spread of malignant tumors accounts
for the majority of cancer- specific deaths
• Therefore possible correlations between EMT markers
and patient prognosis have been intensely studied in
multiple tumor entities.

112. • In colon cancer
• Upregulation of genes involved in EMT/matrix
remodeling defines a molecularly distinct subtype
with very unfavorable prognosis.
• Downregulation of E-cadherin in patient samples,
seems to be associated with high TNM stages and
distant metastasis.

113. •In prostate cancer
• Levels of Twist and Vimentin assessed by
immunohistochemistry in radical prostatectomy
specimens
• Are independent predictors for biochemical
recurrence
• Correlate with resurgence in serum prostate-
specific antigen (PSA) levels following surgery.

114. • Additionally, loss of membraneous E-cadherin
staining is associated with
• Increased Gleason score.
• Advanced clinical stage.
• Poor prognosis in prostate cancer.
• In basal-like, triple-negative breast cancers
• Upregulation of Vimentin associated a poor prognosis.

115. EMT in therapy resistance
• EMT-like cellular phenotype in both surgical
specimens and cell lines is associated with
increased resistance to most conventional
approaches
• Chemotherapy
• Radiotherapy
• Hormone withdrawal

116. • In non-small cell lung cancer cells
• Mesenchymal-like cells (that express Vimentin or
Fibronectin) are less sensitive to EGFR kinase
inhibitors.
• Also in urinary bladder, head and neck, pancreas, and
colorectal carcinoma
• An EMT-like phenotype of the tumor cells is
associated with resistance to anti EGFR therapy.

117. • The resistance to antineoplastic therapies:
• Might be due to stem-cell like properties of tumor
cells that have undergone EMT
• Allowing for self-renewing of a proportion of cells
within the tumor based on the activation of
central signaling pathways. ( Wnt, Notch etc)

118. EMT as potential target for
antineoplastic therapies
• Since the population of stem cell-like tumor cells will
always bear considerable resistance to conventional
therapies efforts have been made to develop
antineoplastic therapies that directly target EMT.

119. Current therapeutic approaches aiming at EMT
Tumors Target Drug Mechanism Effect
Breast LYN Kinase Dasatinib
Metformin
Transcriptional
repression
- Invasion
+E Cadherin
Urothelial uPA Sorafenib Inhibition of
MAPK signalling
-uPA
+E Cadherin
Hepatocellular ILK Kinase activated
ILK
( S343A)
Reduction of
AKT signalling
Increased
senstivity to anti
EGFR therapy
Pancreatic Hedgehog genes Cyclopamine
Resveratol
Transcriptional
repression
-Metastasis
+E Cadherin
Lung HAT/HDAC Vorinostat
Romidepsin
+HAT
-HDAC
Repression of
EMT genes

120. In the era of molecular pathology a good understanding
of EMT and metastasis is essential to better patient
management.

122. THANK
YOU
Next activity (16.10.17)- Lecture by:
Dr Vijay