SHINING A LIGHT ON THE GENOME’S ‘DARK MATTER’
NON-CODING RNAs (nc RNAs) : MICRORNAs AND CANCER
MICRORNAs (MiRs) AND CANCER
Outline of Presentation
• Non-coding RNAs (ncRNAs) and microRNAs
(MiRs)-background and functions
• MiR expression in tumors and cancer cells
• MiRs as prognostic factors for cancer patients
• miRs in body fluids and their potential as
prognostic and diagnostic biomarkers
• Examples of miR functions in cancer
•MiRs as drug targets
ncRNAs IN BACTERIAL AND
EUKARYOTIC GENOMES % OF ncRNAs*
1.4%
29%
27%
2
3
20%
70%
86%
91%
92%
4
5
6
7
8
100%
50%
0%
1
1 Homo Sapiens
2 Arabidopsis thaliana
3 Caenorhabditis elegans
4 Drosophila melanogaster
5 Saccharomyces cerevisiae
6 Escheichia coli
6 Mycobacterium tuberculosis
7 Archaeoglobus fulgidus
* From Szymanski and Barciszewski Genome Biol.3, 005.1, 2002
nc RNAs – “Dark Matter” Function
nc RNA*
small
(20-300 nt)
microRNAs
(~23 nt)
Piwi-interacting
RNAs (piRNAs,
26-31 nt)
Promoterassociated RNAs
(small/variable)
• decrease
mRNA stability/
translation
• gene silencing/
methylation
• gene silencing..?
many more
long
(>200 nt)
Long intergenic
non-coding
RNAs (ncRNAs)
• chromatin
association and
gene modulation/
silencing
* Bioessays 32, 599, 2010; Cancer Res 71, 3, 2011
MicroRNA REGULATION OF GENE
EXPRESSION IN CANCER CELLS/TUMORS
miR-27a
miR
20-22 nt
Pre-miRNA
miR-27a
RNA
(single chain)
• miRs interact with 3’-UTR of mRNAs
• Low miR-mRNA base specificity
(6-8)
• Each miR can potentially interact
with several hundred mRNAs
• Function: block gene expression
COMPLEXITIES OF MiR-mRNA INTERACTIONS
MULTIPLE MiRs REGULATE A SINGLE mRNA*
• the p21 3’UTR can potentially be targeted by
266 miRs (p21 – tumor suppressor)
266-miRs
Transfected
luc
3’UTR
p21
HEK293 cells
“Oncogene 29, 2302, 2010”
• 28 miRs interacted with
3’-UTR; decreased
luciferase activity
• overexpression of miRs
decreased p21 protein
and mRNA levels
MicroRNA ACTIVITY IN CANCER: TUMOR
SUPPRESSIVE OR ONCOGENIC
miR
Tumor suppressive
miR
Oncogenic
• Suppress expression of
oncogenes, growth promoting,
survival and angiogenic genes
(low in tumors)
• Suppress expression of
tumor suppressor, growth
inhibitory, proapoptotic genes
(high in tumors)
INDIVIDUAL MiRs ASSOCIATED WITH
MULTIPLE TUMORS
miR
TS/OG
Tumors
Let-7 Family
TS
10
MiR-159/16-1 cluster
TS
7
MiR-17-92 cluster
OG
7
MiR-26a
TS/OG
4
MiR-34a/b/c
TS
6
MiR-21
OG
 10
TS = tumor suppressor; OG = oncogene
SPECIFICITY OF MiR EXPRESSION IN TUMORS*
miR
Tumor Tissue
TS/OG
MiR-155
Hematopoietic system
OG
MiR-200/141 family
Epithelial-specific
TS/OG
MiR-205
Epithelial-specific
TS/OG
MiR-206
Skeletal and muscle
TS
MiR-9
Nervous system
TS/OG
TUMOR-SPECIFIC PATTERNS OF MiR EXPRESSION
LIVER CANCER-CELL GROWTH/APOPTOSIS*
DOWNREGULATED
let7
miR-1
c-Myc Bcl-XL c-Met FoxP1
miR-122
miR-124
cyclin SRF
G1
CDK-6
miR-199
miR-283
mTOR c-Met cyclin
D2
cyclin
E2
UPREGULATED
miR-101
McL-1
FOS
miR-101
cyclin
D1
CDK6
let7
multiple
miR-106b-25
p21
miR-21
PTEN
miR-2a
miR-602
YAP
RASSFLA
*Br.J.Cancer 104, 235, 2011
(partial list)
Bim
miR-124
p27
p57
miR-18a
ERa
miR-101
APL-5
TUMOR-SPECIFIC PATTERNS OF MiR EXPRESSION
LIVER CANCER-CELL GROWTH/APOPTOSIS*
miRs
• Corresponding
growth promoting/
prosurvival
mRNAsupregulated
miRs
• Corresponding
growth
inhibitory/antisurvival mRNAsdownregulated
CORRELATION OF MIR EXPRESSION WITH
PROGRESSION AND PROGNOSIS OF GASTRIC CANCER*
PATIENTS: 181 patients from 2 cohorts (Japan)
CLASSIFICATION: Stages I-IV
Diffuse vs. Intestinal type
ANALYSIS: • Custom miR microarray chip (Ohio State Univ.)
• miR expression in 160 paired samples
(tumor vs. non-tumor)
• Correlations of miR expression vs. stage,
type and prognosis (survival)
* Lancet Oncol. 11,136, 2010
GASTRIC CANCER MiR SIGNATURE*
UPREGULATED MIRs (22): miR-181 (6), miR-21, miR-25,
miR-92 (2), miR-19b (2), miR-17-92 (7), miR-224, miR-19a
miR-345, miR-191, miR-135b, miR-135a (2)
DOWNREGULATED MIRs (13): miR-148 (2), miR-375,
miR-29b (2), miR-29c, miR-152, miR-218-2, miR-451,
miR-30a-d (5), miR-422b
Several different miRs in the cluster. Lancet Oncol. 2010
MiRs AS PROGNOSTIC FACTORS:
GASTRIC CANCER SURVIVAL*
Intestinal-Type Gastric Cancer
miR-495
HAZARD RATIO
(disease free survival)
5
4
3.2
3
2
1
HAZARD RATIO
(disease free survival)
10
9
8
7
6
5
4
3
2
miR-199
Let-7g
1
0
0
Stages
I-II
Stages
III-IV
high low
I-II
III-IV
high low
I-II
III-IV
low high
I-II
III-IV
SERUM AND BODY FLUID MiRs AS
BIOMARKERS
• Multiple miRs have been characterized not only in
serum but also tears, urine, breast milk, seminal
fluid, saliva, amniotic fluid, bronchial lavage,
cerebrospinal fluid, pleural fluid, peritoneal fluid
and colostrum
(Clin. Chem 56, 1733, 2010)
• A select number of miRs may serve as diagnostic
markers for different tumor types
(Mol. Cancer 9:306, 2010)
SERUM MiRs AS MARKERS FOR LIVER
PATHOLOGIES
(Clin. Sci 120, 183, 2011)
Serum
MiR-885-5p Levels
Rel. miR-885-5p Expr
10
8
6
4
MiR-885-5p
• Elevated in sera
from patients with
liver pathologies
• Primarily
expressed in liver
2
Normal
HepatoLiver
cellular
cirrhosis
Carcinoma
• Function - not
known
MiRs AS PROGNOSTIC FACTORS FOR
CANCER: SUMMARY
• There are unique miR signatures for different cancers
• Several miRs are up-or downregulated in multiple
tumors
• Tumors and serum miR expression can be prognostic
factors for patient survival
• Since multiple miRs target unique and overlapping
mRNAs, are there functional individual miRs and can
they be targeted by anticancer agents?
EXAMPLES OF FUNCTIONAL MiRs IN
TUMORS
miR-21 • Highly expressed in multiple tumors
• Modulates expression of apoptotic/growth
inhibitory mRNAs
miR-17-92 • Highly expressed cluster in multiple tumors
• Several paralogs (same seed sequence)
modulate expression of anti-carcinogenic
mRNAs
miR-335
• Low expression in tumors
• Expression studies suggest that miR-335
inhibits metastasis
MiR-21 IS ONCOGENIC IN VITRO
• Multiple studies show that knockdown of
miR-21 in cancer cells decreases growth
and induces apoptosis
• Overexpression of miR-21 in cancer cells
enhances their tumorigenicity
• miR-21 is a prognostic factor for poor
patient survival
• miR-21 also plays a role in drug-resistance
MiR-21 IS A NEGATIVE PROGNOSTIC FACTOR
FOR PANCREATIC CANCER PATIENTS*
100
low miR-21
Radically resected
Overall Survival (Mo)
% of patients
high miR-21
50
0
Grade
20
metastatic
0
1-2
3
(1000X higher in tumor
vs. non-tumor tissue)
• Patients were
treated with
gemcitabine
• miR-21 also
linked to
gemcitabine and
drug resistance
(5-FU)
*Cancer Res 70, 4528, 2010; PLOS One 5, e10630, 2010
ONCOMIR-21: IN VIVO TRANSGENIC MICE
OVEREXPRESSING MIR-21
(Nes Cre 8, miR-21LSL-Tetoff) (DOXYCYCLINE )
Mice with lymphomas (+) Doxycycline
Survival (%)
100
(-) Doxycycline
Age (days)
100
• Overexpression
of miR-21 in mice
results in pre-B
malignant
lymphoid tumors
• Doxycyclineinduced
downregulation
of miR-21 inhibits
oncogenesis
ONCOMIR-21: IN VIVO KNOCK OUT STUDIES
Lung Tumors
Adenoma
3
Lesions/Lung
Rel. Tumor Areas
20
10
0
K-RasLA2 K-RasLA2
miR-21-/-
• No obvious
phenotype in miR21-/- mice
• Decreased DMBAinduced skin
cancer (miR-21-/-)
0
K-RasLA2 K-RasLA2
miR-21-/-
*Cancer Cell 18, 282, 2010
PNAS 108, 10144, 2011
• miR-21-/suppresses K-ras
induced lung
cancer
REGULATION OF SPECIFICITY PROTEIN (Sp)
TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS)
•Low expression
in non tumor
tissue
• High expression
in tumor tissue
REGULATION OF SPECIFICITY PROTEIN (Sp)
TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS)
Mechanisms for Sp overexpression
• Epigenetic effects (hypo/hypermethylation)
-no evidence
• Enhanced expression of genes that regulate Sp TFs
-they are self regulatory
• Inhibition of “Sp repressors”……..by miRNAs?
REGULATION OF SPECIFICITY PROTEIN (Sp)
TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS)
MiR-27a : ZBTB10?
LAQ824
SKBR3
(HDACi)
CELLS
as-MiR-27a
5 microRNAs (miRs) +
5 hr
22 miRs (including miR-27a)
ZBTB10 + (1.4 Fold)
miR-27a
Note:
ZBTB10
mRNA
Cancer Res. 66, 1277,2006
One of several
hundred potential
miR-27a target
REGULATION OF SPECIFICITY PROTEIN (Sp)
TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS)
ZBTB10 – an Sp repressor
•
ZBTB10 competitively binds (and displaces Sp)
GC rich sequences (JBC 274, 8123, 1999)
• ZBTB10 is a member of the BTB/POZ family of
transcription repressors
• Does miR-27a repress ZBTB10 and thereby allow
for overexpression of Sp1, Sp3 and Sp4?
REGULATION OF SPECIFICITY PROTEIN (Sp)
TRANSCRIPTION FACTORS BY MiRs (ONCOMIRS)
High Basal Sp Expression
in Tumors
miR-17-5p
miR-27a
miR-20a
(a)(a)
Cancer
Res. 67
Oncogene
11011,2012
2007
LOW
HIGH
ZBTB10
ZBTB4
ZBTB10
ZBTB4
EGFR,
CD1,
c-Met
(growth)
Sp
Sp
GC
low
VEGF,
VEGFR1
VEGFR2
(angiogenesis)
Sp
bcl-2,
survivin
NFkB,
p65
(survival) (inflammation)
SUMMARY OF ONCOMIRS THAT SUPRESS Sp
TRANSCRIPTION FACTORS
caspases (active)
proteasomes
ROS
miR-20a
miR-27a
phosphatases
Sp- regulated genes
mitochondria
Sp-repressor
(mRNA)
ZBTB10
ZBTB4
EGFR, CD1,
c-Met (growth)
VEGF,
VEGFR1/VEGFR2
(angiogenesis)
bcl-2, survivin
(survival)
NFkB/p65
(inflammation
Mechanisms of drug-induced repression of Sp1, Sp3, Sp4 and Sp-regulated
genes in cancer cell lines (curcumin, celastrol, aspirin, betulinic acid….)
DEVELOPMENT OF ANTICANCER
DRUGS THAT TARGET MiRS
• Several anticancer drugs
downregulate miRs and induce Sp
repressors which downregulate Sp1,
Sp3 and Sp4 genes.
• Can anticancer drugs induce tumor
suppressor-like miRs ?
2,3,7,8-TCDD AS AN ANTIESTROGEN
AhR
Cl
O
Cl
Cl
O
Cl
arnt
TCDD (
)
arnt
ER
ER
ERE
AhR
DRE
Estrogenic Responses
Inhibited by AhR-ER Crosstalk
• Mammary tumor formation
and growth (rodent & human)
• Uterine and endometrial
responses (rodents)
• Breast cancer cell
responses
Ah RECEPTOR AS A DRUG TARGET
*Toxic responses
(chloracne, wasting…)
Drug
AhR
arnt
*Biochemical
responses (CYPlA,
UGT/GST…)
DRE
*Age, sex, species, strain
and tissue-dependent
*Pharmacolologic
responses
(antiestrogenicity,
anticancer activity,
autoimmune diseases)
DEVELOPMENT OF NON-TOXIC
AhR-BASED ANTIESTROGENS
CH2OH
O
N
H
1,3,6,8-
Indole-3-carbinol (I3C)
CH2
O
2,4,6,8Alternate-substituted
Alkyl PCDFs (synthetic)
N
H
2
Diindolylmethane (DIM)
PROPERTIES OF ALKYL PCDFs 6-MCDF*
• moderate AhR binding
affinity
• low toxicity and poor
induction of CYP1A1
Cl
Cl
O
CH3
Cl
• exhibits partial AhR
antagonist activity (for
toxic responses)
6-MCDF
• but elicits high antiestrogenic activity in
• MCF-7 cells/rat uterus (agonist activity)
* Selective AhR modulator (SAhRM)
SAhRMs FOR BREAST CANCER
THERAPY
• MCDF alone or plus tamoxifen are highly effective
against ER+ breast cancer
(Cancer Res 61, 3902, 2001)
• MCDF inhibits ER- breast cancer cell and tumor
growth
(Endocr Rel. Cancer 16, 835, 2009)
• Inhibition by MCDF not related to altered kinases,
apoptosis or cell cycle genes
• Do SAhRMs such as MCDF work through miRs ?
INDUCTION/REPRESSION OF MiRs BY
TCDD/MCDF
Repressed MiRs
Induced MiRs
Let-7d
miR-134
miR-198
miR-373
miR-126
miR-205
miR-335
INDUCTION OF POTENTIAL TUMOR
SUPPRESSOR MiRs BY TCDD/MCDF –MiR-335*
• Low expression of miR-335 in breast
cancer predicts poor metastasis-free
survival.
• Knockdown of miR-335 enhances MDAMB-231 metastasis whereas
overexpression of miR-335 blocks
metastasis
• miR-335 suppresses expression of
“prometastatic” genes such as SOX-4
* Massague et al Nature. 451, 177, 2007
TCDD/MCDF INDUCE MiR-335 IN MDA-MB-231
CELLS - AHR-DEPENDENT
MDA-MB-231
miR-335
4.5
4
MDA-MB-231
miR-335
5
TCDD


4
3.5
3


TCDD
MCDF
3
2.5

2
Fold Induction
Fold Induction
AhR
Actin
iCT
iAHR
MCDF
iCT iAhR

1.5
1
0.5
0
2
1
0
0 hr
12 hr
24 hr
DMSO
TCDD AND MCDF INHIBIT BREAST CANCER CELL
MIGRATION AND INVASION-BOYDEN CHAMBER ASSAY
MDA-MB-231/24hr
120
100
Upper
Chamber
*
Lower
Chamber
Cells
Membrane
% Control
80
*
60
40
20
Pores
Migrated
Cells
0
CTL
5
10
MCDF(uM)
MIR-335 ALSO INDUCES CELL INVASION BUT THIS
RESPONSE IS AHR-INDEPENDENT
Invasion Assay/MDA-MB-231
120
iCTL
100
iAhR
% control
80
**
60
iCTL+CTL
iCTL+miR335
40
20
0
CTL
miR335
iAhR+CTL
iAhR+miR335
TCDD/MCDF DECREASE MIR-335-REGULATED
SOX4 IN MDA-MB-231 CELLS
1.2
1.6
DMSO
TCDD
SOX4 mRNA (Rel. DMSO)
SOX4 mRNA (Rel. DMSO)
1.4
1.2
1
0.8
*
0.6
0.4
0.2
DMSO
MCDF
1
0.8
0.6
*
0.4
0.2
0
0
iCTL
iAhR
iCTL
iAhR
TCDD/MCDF DECREASE SOX4 PROTEIN IN
MDA-MBA-231 CELLS
MDA-MB-231
iAhR
DMSO
TCDD 10 nM
MCDF 5 nM
SOX4
β-Actin
+
-
+
-
- + + +
- + - - - + + - - +
MCDF INHIBITS MDA-MB-231 CELL LUNG
METASTASIS IN MICE
No. of colonies
100
MCDF
(40 mg/kg/d)
80
No. of colonies
Corn Oil
(CTL)
60
40
ND
20
0
1
No cells
CO
2
+
T
MCDF
3
+
T
ANTIMETASTATIC ACTIVITY OF AHR AGONISTS
IN ER BREAST CANCER
Ligand
activated
Ahr
AhR
arnt
miR-335
Normal cells
Preneoplastic
miR-335cells
Cancer cells
(Invasive carcinoma)
Metastasis
SOX4
SOX4 and other miR-335
regulated metastatic
mRNAs
SOX4
SOX4 and other miR-335
regulated proteins
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