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Tumour invasion and metastasis initiated by miRNA-10b in breast cancer. Ma et al Erin White 9-18-08. miRNAs. miRNA regulate genes via imperfect complementarity to 3’ UTR of gene Cleaved by Dicer from 70-100nt pre-miRNA hairpin in cytosol Can be expressed in a tissue specific fashion.

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tumour invasion and metastasis initiated by mirna 10b in breast cancer

Tumour invasion and metastasis initiated by miRNA-10b in breast cancer

Ma et al

Erin White

9-18-08

mirnas
miRNAs
  • miRNA regulate genes via imperfect complementarity to 3’ UTR of gene
  • Cleaved by Dicer from 70-100nt pre-miRNA hairpin in cytosol
  • Can be expressed in a tissue specific fashion
mirnas expressed in breast cancer
miRNAs expressed in breast cancer
  • miR-155
  • miR-9 highly expressed in met. cancer
  • miR-10b
  • miR-335
  • miR-206 low expression in met. cancer cells
  • miR-126
mir 10b positively regulates cell migration and invasion
miR-10b positively regulates cell migration and invasion

Inhibition of miRNAs

to determine regulation

of migration/invasion.

Comparison of normal

mammary cells to

tumorigenic but non-met

cells.

mir 10b induces tumour invasion
miR-10b induces tumour invasion

Tumors produced

in NOD-SCID mice

after 11 wks.

Muscular

H & E staining of

tumors after 6

wks. Tumors

expressing miR-10b

are invasive.

Vascular

mir 10b induces tumour invasion8
miR-10b induces tumour invasion

Ki-67 and MECA-32

staining of mammary

tumors in NOD-SCID

mice marking

proliferation and

epithelial cells.

Invasion fronts of

miR-10b express’g

tumors show high

levels of cell

proliferation and

angiogenesis

mir 10b induces distant metastasis
miR-10b induces distant metastasis

Highly prolif.

cells in lumina

of vessels

associated

with miR-10b

tumors.

Micro-met in

lung sections

after 9 wks.

Cytokeratin

staining.

mir 10b induces distant metastasis10
miR-10b induces distant metastasis

Lungs from mice

receiving miR-10b

injexn after 11 wks.

twist and snai1
Twist and Snai1
  • Transcription factors
  • Twist is a metastasis-promoting gene
  • Epithelial-Mesenchymal transition (EMT) inducing factors
  • Authors think that Twist might regulate miR-10b expression
  • Snai1 used as a control in assays
mir 10b is regulated by twist
miR-10b is regulated by Twist

Expression of miR-10b in

four mouse mammary

tumour cell lines with

Twist as regulator

Putative

transcription factors

regulating miR-10b

Putative binding sites

for transcription

regulation in miR-10b

mir 10b is regulated by twist13
miR-10b is regulated by Twist

ChIP assay to determine where

Twist binds miR-10b gene

Overexpression of Twist leads to

increase in motility and invasiveness

5-fold decrease by miR-10b inhibition

hoxd10
HOXD10
  • Homeobox domain 10
  • Previously implicated in suppression of cell migration and/or invasion
  • Expression lost in malignant breast cancer
  • Restored expression in MDA-MB-231 cells impairs migration and invasion in vitro and in vivo
  • 3’ UTR has partial complementarity to miR-10b
slide15
RHOC
  • Involved in cell migration and ECM remodelling
  • Identified as important player in metastasis
  • Robustly expressed in miR-10b-transduced cells
  • Repressed by HOXD10
mir 10b suppresses hoxd10 leading to induction of rhoc
miR-10b suppresses HOXD10, leading to induction of RHOC

miR-10b does not

cause degradation

of HOXD10 mRNA

Reduction in activity

of 3’ UTR due to

binding of miR-10b

HOXD10 protein levels

reduced by miR-10b

expression as RHOC

increases

mir 10b suppresses hoxd10 leading to induction of rhoc17
miR-10b suppresses HOXD10, leading to induction of RHOC

Constitutive

expression of

HOXD10 ablates

cell migration

and invasiveness

Knockdown of

RHOC reduces

invasion and

migration

conclusions
Conclusions
  • miR-10b is highly expressed in metastatic breast cancer cells
  • miR-10b induces tumour invasion
  • miR-10b induces distant metastasis
  • miR-10b is regulated by Twist transcription factor
  • miR-10b suppresses HOXD10
endogenous human mirnas that suppress breast cancer metastasis

Endogenous human miRNAs that suppress breast cancer metastasis

Tavazoie et al

Erin White

9-18-08

cell lines used22
Cell Lines Used
  • Derivatives of MDA-MB-231 parent line
    • 231 cells poorly metastatic, high miR-335 expression
    • LM2 = highly metastatic to lung tissue
    • BoM2 = highly metastatic to bone
  • CN34 = cells derived from patient with metastatic breast cancer
    • CN34-LM1
    • CN34-BoM1
id of mirnas that suppress lung and bone metastasis
ID of miRNAs that suppress lung and bone metastasis

Lung sections after 8 wks

showing decrease in number of metastastic foci

miR-122a restoration did not result in signif. decrease in lung colonization

Restoration of miR-335, -206, and -126 decreased lung colonization by five-fold

id of mirnas that suppress bone and lung metastasis
ID of miRNAs that suppress bone and lung metastasis

Expression of miR-335, -206, and -126 decrease bone metastasis

id of mirnas that suppress lung and bone metastasis25
ID of miRNAs that suppress lung and bone metastasis

Mice injected with

cells from pleural

fluid of patient with

met breast cancer

miR-335, -206, and -126 reduced the ability of CN34-LM1 and CN34-BoM1 cells

to metastasize to lung and bone

Low levels of suppressor miRNAs shown in CN34 cells

clinical association of mir 335 126 with metastasis free survival
Clinical association of miR-335, -126 with metastasis-free survival

Median expression values of these miRNAs was eight-fold lower in

patients that did relapse compared to those who did not relapse.

mir 335 regulated gene set includes sox4 as direct target
miR-335-regulated gene set includes SOX4 as direct target

3’ UTRs of all genes in set

have complementarity to

miR-335. Activity assayed

via luciferase in LM2 or 231

cells

Inhibition of miR-335 in 231 cells

enhanced expression of gene set

sox4 is a mir 335 direct target
SOX4 is a miR-335 direct target

miR-335 suppresses activity of wt SOX4 3’ UTR but mutation abrogates suppression

mir 335 regulates metastasis and invasion through suppression of sox4 and tnc
miR-335 regulates metastasis and invasion through suppression of SOX4 and TNC

Knockdown of

SOX4 or TNC

reduces the

invasiveness of

LM2 cells

metastasis free survival of patients expressing mir 335 six gene signature
Metastasis-free survival of patients expressing miR-335 six-gene signature

Patients whose primary breast tumors were positive for metastasis signature had

worse metastasis-free survival

conclusions33
Conclusions
  • miR-335, -206, and -126 are suppressors of metastasis
  • miR-335 and miR-206 regulate migration, invasion, and morphology
  • miR-126 suppresses overall tumour growth
  • miR-335 regulates a set of metastasis genes including SOX4 and TNC as direct targets