Tugas Biochemistry II

8/20/2019 Tugas Biochemistry II

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疾患制御学特論

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博士前期課程１年

21261231LIA HAFIYANI


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（Original article is attached in the end of this review）


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Focal Adhesion Kinase (FAK is a focal adhesion-associated protein kinase involved in cellularadhesion and spreading processes. It has been shown that when FAK was blocked, breast cancer cellsbecame less metastastic due to decreased mobility!"#$2 is a non-receptor tyrosine kinase of the FAK family, predominantly expressed in the cells derivedfrom hematopoietic lineages and in the central nervous system. It is involved in calcium inducedregulation of ion channel and activation of the map kinase signaling pathway. It plays an important rolein cell motility such as spreading and migration."F%22& ' FAK spesic inhibitor"F 21 ' FAK!"yk# inhibitor

$etastasis suppressors alter the signal transduction of cancercellsPatricia S. Steeg%ature &eviews 'ancer (, ))-*(

A schematic of the metastatic process, beginningwith a + an in situ cancer surrounded by an intacbasement membrane. b + Invasion reuiresreversible changes in cellcell and cellextracellular-matrix adherence, destruction oproteins in the matrix and stroma, and motility$etastasiing cells can c + enter via thelymphatics, or d +directly enter the circulation. e /urvival and arrest 0detach1 of tumour cells, andextravasation of the circulatory system follows. f $etastatic coloniation of the distant site

progresses through single cells, which mightremain dormant for years, to establish tumomicroenvironment and occult micrometastasesand g + progressively growing, angiogenicmetastases./o we have seen that the ability of the cells toinvade through the lamina basalis can only occuif there is a reversible changes in cell and cell andcell-extracellular matrix, destruction of stromaand matrix, and motility. 2he important programto facilitate this process is what we3ve known as4$2, or epithelial mesenchymal transition, whichis the process whereby immotile, polaried

epithelial cells transition into highly motile, apolabroblastoid-like cells.

2he "athophysiology of 4pithelial-$esenchymal 2ransitionInduced by 2ransforming 5rowth Factor-6 in %ormal and$alignant $ammary 4pithelial 'ellsMolly A. Taylor & Jenny G. Parvani & William P. Schiemann 7 $ammary 5land 8iol %eoplasia 0#9:91 :);:*


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"#$2/tructurally similar to FAK, but they exhibit some important dierences ; FAK is ubiuitous, essential for lifeand localies primarily to focal adhesion complexes where it is activated by growth factor receptors. "yk#is highly restricted, its expression is dispensable for life. 2he other dierence also that "yk# is involved incalcium induced regulation of ion channel and activation of the map kinase signaling pathway.Although elevations in the expression and activity of FAK are clearly associated with breast canceprogression and metastasis, a similar understanding related to the function of "yk# during mammary

tumorigenesis remains to be elucidated. Bowever, the expression and activity of "yk# has also beenimplicated in regulating the production and expansion of breast cancer stem cells, as well as in promotingthe motility and survival of breast cancer cells. Berein, we identify "yk# as a novel 4$2 biomarker andfound its expression to be robustly upregulated by 25F-b in transdierentiating breast cancer cells. 2hus,the obCective of this study was to determine the mechanism whereby "yk# contributes to breast cancermetastasis.As such, they performed Dncomine ?.? analyses and observed "yk# expression to be signicantlyupregulated in B-&as-transformed mammary epithelial cells, and in invasive human breast carcinomasparticularly those that exhibited recurrence 02able :1. $oreover, "yk# expression was signicantly reducedin human $@A-$8-#(: 0$@A-#(:1 metastatic breast cancer cells engineered to lack expression of 'oco,#)a bone morphogenetic protein antagonist 05/4#E9?


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limited but signicant lung metastatic activitycompared with the parental population. =hen:E(?-derived lung lesions were expanded inculture and reinoculated into mice, these cells0denoted J$: subpopulations, showed increasedlung metastatic activity. Another round of selection in vivo yielded second-generationpopulations 0denoted J$#1 that were rapidly andeLciently metastatic to the lungs. Bistologicalanalysis conrmed that J$# lesions replacedlarge areas of the lung parenchyma, whereas:E(? cells exhibited intravascular growth withless extensive extravasation and parenchymal

involvement. Inoculation of as few as # M :9(J$# cells was suLcient for the emergence ofaggressive lung metastases, whereas inoculationof # M :9) parental cells left only a residualindolent population in the lungs. Furthermore, theenhancement in lung metastatic activity wastissue specic. =hen J$# populations wereinoculated into the left cardiac ventricle tofacilitate bone metastasis, their metastaticactivity was comparable to that of the parentaand :E(? populations, and it was markedlyinferior to that of a previously described, highlyaggressive bone metastatic population.

Fig,re 1! .levated "#$2 e/-ression and activit# is a -otential *ediator of *etastatico,tgrowth +# +reast cancer cells

A. 2hey monitored "yk# expression levels in

parental and clonal cell populations o

$@A-$8 #(: cells, which represent a

heterogenous mixture of aggressive andnon-aggressive carcinoma cells. FAK

expression was euivalent across all clona

cells, "yk# is highly variable and

dissociated, from dierential invasiveness8. Bere we can see that "yk# expression was

inversely correlated with the invasive

potential of $8 #(: cells.'. "yk# expression is positively associated

with the competency of $8 #(: cells to

thrive in (@-organotypic cultures 0that

mimicked the pulmonary

microenvironment1.@. Joosely packed and branched organoid

structures linked to the development of

metastatic dormancy 0in previous

research1. Administration of dual FAK!"yk#

inhibitor, signicantly more eective a

inhibiting the (@ growth.

Fig,re 2! 0)F%+ ind,ces "#$2 e/-ression via *ad% and rc%de-endent -athwa#s


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A. 25F-b induced "yk# expression in $8 #(:

cells in traditional #@-cultures, andmagnitude of "yk# expression potentiated

by using (@ organotypic cell cultures.8. Bere they use # types of murine breast

cancer cell lines, highly metastatic ?2:

and less aggressive counterparts ?29N,

where "yk# is expressed higher in ?2:

A%@ signicantly upregulated when

stimulated by 25F-b, in time dependent

manner. 0=hile FAK expression is not

correlated with this metastatic ability1

&eal time "'& 0m&%A level1.'. Idem with 8, using immunoblotting.@. 2b&: and src inhibitor supressed the

expression of "yk# , implicating autocrine

25F-b signalling and its elevation of "yk#

is driven by /rc. 0they already conduct the

same procedures with anothe

noncanonical 25Fb eector molecules

$4K:!#, $A"K, AK2, /2A2,IKK, dll but with

negative result1.4. 2he activation of /mad#!( by 25F-b was

abrogated upon propagating cells in

compliant (@ organotypic cultures

condition that elicited "yk# expression

independent of 25F-b signalling

$oreover, by inhibiting src not only

decreased the ability of 25F-b to induce"yk#, but AJ/D the phosphorylation o

/mad#!( 0supp gure #d and (b1. this

show the role of coupling 25F-b and "yk#

expression in the signalling of /mad #!(.F. @epletion of smad? abrogated the

coupling of 25F-b to "yk# expression.

Fig,re 3! "#$2 e/-ression is essential for -,l*onar# t,*or for*ation in *ice

A. 2hey depleted "yk# expression in highly

metastatic ?2: cells.8. 4ngrafted onto the mammary fat pads o

8alb!' mice. "yk# deLciency failed to

aect the growth and latency of primary

?2: tumors.'. 8ut its impair the extent and sie to the

disseminated ?2: cells formed

macroscopic pulmonary metastases. 0)

weeks after engraftment, mice were

euthanied and pulmonary metastatic

nodules were uantied1.

@. &epresentative lungs and the small focfrom pyk# deLcient cells.

Fig,re ! "#$2 e/-ression is essential for -,l*onar# t,*or for*ation in *ice


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A. 2hey use ?29N cells, orthotopic cancer cells can be detected in the circulatory system but failed toestablish proliferating secondary tumors. 8ut when the cells are inCected in the lateral vein of the

tails, ist exhibit aggressive coloniation of multiple tissues, including brain 0data not shown1, lung

and various skeletal sites. 0bioluminescence18. "yk# expression was uniformly upregulated in ex vivo cultures of ?29N metastases as compared

with the parental counterparts.'. 2hey use ?29N cells deLcient in "yk# expression 0sh"yk#1 and pictures shows signicant reduction

in the growth of secondary tumor lesions in the lung and other organ sites. 0bioluminescence1.@. @ata are mean area >ux values normalied to inCected values, after ( weeks.4. "yk# deLcient cells also greatly extended the overall survival of tumor bearing mice.

F!

)! Fig,re ! "#$2 e/-ression is dis-ensa+le for +reast cancer cell invasion sti*,lated +#0)F%4H!

I! 5!K!L!!A. 2hey use static micrographs, to show tha

25F-b treatment of ?29N cells resulted in

robust rearrangement of actin cytoskeleta

system. Interestingly, administration o

both inhibitors 0"F-##E and "F-#N:1 also

elicited accumulation of lopodia and

formation of actin stress bers, but timelapse microscopy 0supp movies1 shows

that these inhibitors inhibit the motility o

the cells by preventing their ability to

turnover the focal adhesion complexes.7! 25F-b induction also enhance the invasion

of ?29N cells, but both inhibitors similarly

failed in induce the invasion phenotypes of

this cells. 2his nding suggest that FAK8

+,t not "#$28 is res-onsi+le for

co,-ling 0)F%+ to +reast cancer cel


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invasion. $eans that if our target is to

alter the invasion ability stimulated by

25F-b, "yk# is not the eective target to

be inactivated.

9! 2o test this hypothesis, they use "yk#

depleted expression in ?29N cells, and

data shows that the invasive response was

similar to the parental counterparts.

N!O!"! Fig,re 6! :-reg,lated "#$2 e/-ression co,-les .0 to *etastatic o,tgrowth sti*,lated

+# 0)F%4

;!

0!:!

=!

>!?! In this @g,re the# want to address that "#$2 ,-reg,lation is a -art of .0 signat,re

o-erant in driving *etastatic o,tgrowth! .0 can circ,*vent +reast cancer dor*anc#and -roliferating -rogra*s8 while "#$2 ena+les disse*inated cells to for* *acrosco-icsecondar# t,*ors in so*e tiss,es!

A. 25F-b stimulation 0post 4$21 signicantly increased the outgrowth of ?29N cells in (@ organotypic

culture, and dual inactivation of FAK!"yk# is more eective in diminishing this outgrowth compared

to inhibition of FAK alone.8. 0also showed the additional information gained by using (@ cultures1 "yk# deLciency did not alte

the #@ morphology of ?29N cells but enhance their formation of branched organoids in (@ culture

a phenotype that associated with mammary gland dierentiation and metastatic dormancy Gin the

previous studyG - phase contrast microscopy.'. "yk# deciency signicantly impair the (@ cultures growth in response to 25F-b 0bioluminescence1.@. 2his bioluminescent images show the importance of upregulated "yk# expression in coupling 25F-b

signaling to the *etastatic o,tgrowth of breast cancer cells.4. Idem @, data are the mean area >ux values.

Y!! Fig,re ! "#$2 is reB,ired for 0)F%4 sti*,lation of an o,tgrowth -roficient -henot#-e

AA!


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A7!

A'.AC! 0he# tr# to deter*ine the

relationshi- +etween "#$2 incoordinating the e/cl,sive e/-ressione/hi+ited +# .%cad and +1%integrin in

o,tgrowth inco*-etent (.%cad high871 integrin low or o,tgrowt-ro@cient (.%cad low8 71%integrinhigh!

A. First, "yk# deciency ?29N cells elicit

upregulation in 4-cad expression. @espite

their elevated basal expression of 4-cad,

"yk# decient ?29N cells remained

competent to downregulate 4-cad in

response to 25F-b. 2hus, unlike FAK, "yk#

is dispensable for general 25F-b signalling

but essential in coupling 25F-b to

b:integrin expression in outgrowth

procient 8''.8. 4ctopic expression of wild-type 4-cad, bu

not A4-4cad prevented 25F-b from

inducing "yk# expression in the

stimulation with 25F-b in a manner that

correlated with the ability of this wild-type

to repress the expression of b: integrin in

metastatic @#.A: breast cancer cells 0in

prev study1.'. =hen propagated in (@ organotypic

cultures, ?29N cells simultaneously

downregulated the expression of 4-cad

and upregulated "yk#. $eans that the

ability of "yk#-decient ?29N cells to

maintain e-cad expression destabilied b:integrin expression, under pulmonary

growth conditions.@. 8: integrin deciency elicited an

upregulation of 4-cad that prevent the

ability of 25F-b and 4$2 to induce "yk#

expression.

A.!AF!A)! Fig,re &! che*atic I*ageAH!

AI! A7. 2aken together from the previous datathis schematic gures shows the dynamicevents reuired for 25F-b and 4$2 toinitiate the pulmonary outgrowth odisseminated breast cancer cells, aprocess that culminating in diminished 4-cad expression, upregulated "yk#expression, and stabilied b:-integrinexpression.


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AK!AL!

A$.

Tugas Biochemistry II

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