Breast remodelling in the reproductive cycle

Question 1

Describe the mammary gland

Answer 1

Ductal tree
Ducts connect to nipple on one end, form lobular alveloi on other
Growth during puberty

Question 2

What regulates growth of the mammary gland

Answer 2

Hormones, GFs, ECM

Question 3

Describe the microenvironment of the mammary gland

Answer 3

Ducts consist of luminal epithelium, myoepithelium + laminin-BM
Luminal epithelium = lactocytes = produce milk
Myoepithelial cells squeeze milk from lactocytes
Stromal collagen + stromal fibroblasts are responsible for producing collagen matrix
Whole structure embedded on fat pad of adipoctyes

Question 4

How does the mammary gland convert into a milk producing organ

Answer 4

Development of alveoli
Lactation differentiation
Cell proliferation + differentiation
Cell polarity

Question 5

What is the role of the basement membrane

Answer 5

Holds cells together and provides signals

Specialised ECM surrounds epithelial tissues + separates them from interstitial stromal collagen compartment

Question 6

What is the composition of the ECM

Answer 6

Laminins 111 + 332
Collagen IV
Nidogen/entactin
Proteoglycans

Question 7

How does cell adhesion to ECM occur

Answer 7

Through transmembrane receptors
Different subunits can cooperate to detect different ECM proteins
B1 integrin important
Integrins bound to ECM on outside, linked to cytoskeleton inside cell
Focal adhesion complex

Question 8

What genetic approaches can be used to study gene function

Answer 8

CRISPR/Cas9
TALEN
short hairpin RNA
Cre-Lox
In vivo tissue specific targeting

Question 9

What does genetic deletion of B1 integrin in mammary gland show

Answer 9

Deleted only from epithelial cells in mammary glands
Reduced alveolar development, reduced proliferation
GFs and hormones can cause phosphorylation of ERK but need signals from integrins which bind to FAK
B1 integrins control cell cycle via Talin + ERK nuclear translocation

Question 10

How does laminin ECM mediate milk protein expression in mammary epithelia

Answer 10

Prolactin binds receptor
Triggers signalling cascade involving Jak2 bound to TF Stat5
Translocates into nucleus to initiate milk production
If the BM is removed them no development so ECM factors required- Laminin

Question 11

How do we know B1 integrin is mediated for lactational differentiation

Answer 11

Pups feeding off transgenic mothers are smaller, mothers have defect- reduced milk fat + milk fat protein
Laminin signals to B1 integrin

Question 12

Is FAK required for lactational differentiation

Answer 12

No
Fak deletion doesnt affect pup weight, milk fat or protein
Not involved in differentiation downstream of integrin

Question 13

What does genetic deletion of ILK show

Answer 13

Pup weights reduced but not as much as B1 integrin

Reduced lactation

Question 14

What does ILK deletion in culture show

Answer 14

Ad-Cre virus removes ILK
Phenotype can be rescued with IL-GFP
ILK is downstream of B1 integrin

Question 15

Describe the lactational pathway

Answer 15

Laminin > B1 integrin > ILK > Rac1 > b-casein

Also: Parvin, a-pix, vinculin

Question 16

What does genetic deletion of Rac1 show

Answer 16

Lobular alveoli much smaller than WT

Not producing as much milk protein

Question 17

What does consitutively active Rac1 do

Answer 17

Rescues lactation defect in integrin/ILK null organoids + MECs on non-permissive collagen I matrix

Question 18

Why is cell polarity important in lumen formation

Answer 18

Hollow lumen essential for proper tissue function

Apicobasal polarisation ensures secretion is directed towards the lumen

Question 19

What does lumen formation look like without B1 integrin

Answer 19

Failed to form lumen

Filled lumens frequent in early breat + epithelial cancers

Question 20

What does deletion of adhesome components show in relation to lumen formation

Answer 20

ILK but not FAK controls lumen formation downstream of b1 integrin

Question 21

What are the hypothetical mechanisms of lumen formation

Answer 21

Apoptotic clearance of cells in the core- not possible, cell death not affected by b1 integrin KO
Controlled cell proliferation- no, KO causes underproliferation
Cell division axis orientation
Cell adhesion- No, cells filling lumen space still have adhesion
Cell rearrangements

Question 22

How is the lumen formed

Answer 22

Apical markers form all over, basal markers move outwards, apical move inwards.
Multiple lumens form then fuse together
B1 integrin required to reorient apical polarity

Question 23

What is the role of B1 integrin in maintenance of polarity

Answer 23

If deleted after polarised lumens form then apicobasal polarity flips
Lumen collapse
Membranes remodelled

Question 24

How is the membrane remodelled at the cell-BM interface

Answer 24

1. Unpolarised cells with fragmented golgi + apical markers form at the membrane
2. Engagement of b1 integrins with BM recruits ILK to instruct orientation of polarity by interacting with plus tips
   Integrin/ILK polarise microtubules along apicobasal axis
   Interactions include endocytosis of apical components
3. Integrins/ILK regulate internal cell polarity by positioning golgi apparatus subapically, aids polarised trafficking to membrane. New apical face on other membrane