MicroRNAs as tumor suppressors and oncogenes Madrid, September 2013

Pedro Medina

14th

ASEICAINTERNATIONALCONGRESSSpanish Society for Cancer Research

Madrid · 23rd-25th September 2013Hospital universitario Ramón y Cajal

6DOXG0DGULG

Hospital UniversitarioRamón y Cajal

Comunidad de Madrid

Overview

Introduction: microRNAs and cancer.

I) let-7 microRNA family as tumor suppressors.

II) mir-21 as an oncogene.

MicroRNAs

• 18-24nts RNAs.

• Post-transcriptional regulators that bind to complementary sequences in the three prime untranslated regions (3' UTRs) of target messenger RNA transcripts (mRNAs), usually resulting in gene silencing.

• Estimates of about a thousand miRNAs in the human genome.

• Each miRNA may control the translation of hundreds of different mRNAs.

• Computational models have indicated that up to 92% of human genes may be regulated by miRNAs. H.G. & W.F. 2008

RISC (RNA-Induced Silencing Complex)

Proto- Oncogenes

Tumor suppressor

Balance

MicroRNAS

Proto-oncogenes > Oncogenes

Tumor Suppressors

Pathological conditions > Cancer

Unbalance

MicroRNAS

MicroRNAs & Cancer

Components of the miRNA machinery and miRNAs themselves are involved in many cellular processes that are altered in cancer, such as differentiation, proliferation and apoptosis. MiRNAs are mutated and/or miss-expressed in cancers Some miRNAs (oncomiRs) regulate key genes in cancer acting as oncogenes and tumor suppressors.

Medina PP & Slack FJ, Cell Cycle 2009

MicroRNAs as oncogenes and tumor suppressors in vivo

I) let-7 microRNA family as tumor suppressors

II) mir-21 as an oncogene.

let-7 miRNA family

•  Lethal-7 (let-7) was discovered originally in C. elegans. •  First microRNA discovered in humans. •  In humans, the let-7 miRNA family is composed by 12 genes, that codes

for 9 mature miRNAs.

Evidence for tumor suppressor function in lung cancer I:

let-7 is expressed at lower levels in lung tumors than normal tissue.

Primary tumors

Northern Blot analysis N: normal T: tumoral.

Non-Lung Lung Cancer

44% of the samples have >80% reduction of let-7 levels

Takamizawa et al, 2004.

Cluster 1: low let-7 levels Cluster 2: high let-7 levels

Significant worse prognosis for patients with reduced let-7 levels.

146 patients

Takamizawa et al, 2004.

Evidence for tumor suppressor function in lung cancer II:

The prognosis in lung cancer patients is directly correlated with let-7 expression levels.

Biological roles of the let-7 miRNA family

Does the let-7 miRNA family function as tumor suppressors in vivo?

Therapeutic value of reintroduction of let-7:

- Lung cancer xenografts (synthetic let-7).

- KRAS G12D lung cancer mouse model (lentivirus let-7).

Does the let-7 miRNA family function as tumor suppressors in vivo?

Therapeutic value of reintroduction of let-7:

- Lung cancer xenografts (synthetic let-7).

- KRAS G12D lung cancer mouse model (lentivirus let-7).

Growth inhibition of established lung tumor xenografts by synthetic let-7 oligonucleotides

Injections

20 days Xenograft

Controls: •  miR-NC: microRNA scramble •  siPORT: vehicle (transfect agent) •  PBS: empty

Lung cancer (H460) xenografts

Trang & Medina et al.

let-7 administration in H460 xenografts reduce cell proliferation and cell viability

v viable cells d non viable cells.

v viable cells d non viable cells.

(scramble microRNA control)

(vehicle control)

(proliferation marker)

Trang & Medina et al. 2010

v viable cells d non viable cells.

IHC RAS

IHC CDC25

Let-7 introduction reduce the mRNA of CDK6 and N-RAS

let-7 targets RAS, CDK6 and CDC25

Trang & Medina et al.

Conclusions

•  Administration of let-7 reduces tumor proliferation and cell viability in H460 xenografts.

•  let-7 inhibits the expression of CDK6, CDC25 and RAS genes.

Does the let-7 miRNA family function as tumor suppressors in vivo?

Therapeutic value of reintroduction of let-7:

- Xenografts (synthetic let-7).

- KRAS G12D lung cancer mouse model (lentivirus let-7).

The let-7 miRNA family inhibits RAS expression (Johnson et al 2005).

Testing ectopic let-7 expression in KRAS G12D lung cancer mouse model

let-7 treatment reduces cell proliferation and tumor burden Cell proliferation

Reduction

Regression

Ectopic let-7 expression reduces cell proliferation and tumor burden

Trang & Medina et al.

Conclusions

• let-7 treatment reduces cell proliferation and tumor burden in KRAS G12D lung cancer mouse model.

let-7 is a tumor suppressor in vivo and the reintroduction of its activity may have a therapeutic value.

MicroRNAs as oncogenes and tumor suppressors in vivo

I) let-7 miRNA family as tumor suppressors

II) mir-21 as an oncogene.

mir-21 has oncogenic functions (cell lines studies): •  Antiapoptotic factor (Chan Jaet al, 2005). •  Invasion (Chan Jaet al, 2005). •  Promoting growth (Si Ml et al, 2007), •  Target tumor suppressors:

• TPM1 (Zhu S et al, 2007). • PTEN (Meng F et al, 2007). • PDCD4 (Frankel, L. B. et al, 2008)

mir-21 & Cancer

To study mir-21 oncogenic properties in vivo

Created a mouse model that over-expresses miR-21:

• Tissue specific (mediated by Cre)

• Inducible (Tet-off system).

Transgenic Mouse to Over-express mir-21: Tissue specific & DOX inducibility (tet-off)

Cre recombinase eliminates the STOP cassette and mir-21 is expressed

Our construct is inserted in the ubiquitously expressed ROSA26 gene

Doxycycline inhibits mir-21 expression

ROSA26 ROSA26 STOP mir-21 Tet-off

DOX

ROSA26 ROSA26 Tet-off mir-21

ROSA26 mir-21

Transcription

LoxP LoxP

Transcription

Cre

ROSA26 Tet-off

DOX administered (Tet-off) mir-21 expression off +  /Nes'n-­‐cre    mir-21LSL-Tetoff /

Nes'n-­‐cre  

No Phenotype No Phenotype

No DOX administered mir-21 expression on +  /Nes'n-­‐cre    mir-21LSL-Tetoff /  

Nes'n-­‐cre  

No Phenotype Phenotype

mir-21 Phenotype: DOX vs NON DOX

2-4 months of age: • Paraparesis (weakness of the movement of the rear limbs) • Lymphadenopathy

mir-21LSL-Tetoff /Nestin-Cre phenotype Paraparesis  (weakness  of  the  rear  limbs)  Lymohadenopathy  

• Enlargement of lymph nodes (lymphadenopathy) • Enlargement of spleen (splenomegalia) • Ruffled fur • Paresis/Paralysis of the rear limbs • Hunched posture • Labored breathing • Ataxia

mir-21LSL-Tetoff/Nestin-Cre Clinical Signs (ranked by prevalence)

Clinical signs fit with leukemia/lymphoma

Phenotype Penetrance

2/3 of the Nestin-Cre/mir-21LSL-Tetoff mice develop lymphadenopathy or paraparesis before 4 months of age. But none of the mice treated with DOX nor the wildtype siblings showed any symptoms

p<0.0001

n=30

n=48 n=69

Hematopoietic organs

Clinical signs fit with leukemia/lymphoma

Splenomegaly    

Bone  marrow  /  lymph    

Thymus  hypertrophy    

650mgr                80mgr  

388  mgr                    2.9  mgr    

     Lymph  adenopathy    

Medina et al. Nature 2010

mir-­‐21                wt  

mir-­‐21                                                wt  

mir-­‐21                            wt  mir-­‐21                            wt  

 What  kind  of  hematological  malignancy  do  our  mice  have?  

Spleen  wt   Spleen  #332  Spleen  #247  

100 101 102 103 104B220-PE

100

101

102

103

104

CD3-FITC

100 101 102 103 104B220-PE

100

101

102

103

104

CD3-FITC

100 101 102 103 104B220-PE

100

101

102

103

104CD3-FITC

CD3-­‐  B220+  

CD3+B220-­‐  (T  cells)  

CD3-­‐B220+  (B  cells)  

B-Cell

Conclusion: B cell lymphoma

CD3: T-cell specific antigen CD45R(B220): B-cell specific antigen

mir-21LSL-Tetoff/Nestin-Cre

Tumor immunotype

CD3

B220

IgM-­‐  B220+  

100 101 102 103 104B220-PE

100

101

102

103

104

IgM-FITC

100 101 102 103 104

FL2-H: PE

100

101

102

103

104

FL1-

H: F

ITC

100 101 102 103 104B220-PE

100

101

102

103

104

IgM-FITC

IgM-­‐B220-­‐  (T  cells)  

IgM+B220+  (mature  B  cells)  

Pre-B-Cell

IgM: Present in mature B Cells CD45R(B220): B-cell specific antigen

Conclusion: Pre-B-Cell Lymphoblastic Lymphoma/Leukemia

Spleen  wt   Spleen  #332  Spleen  #247  mir-21LSL-Tetoff/Nestin-Cre

Tumor immunotype

IgM

B220

Conclusions •  mir-21 mice develop Lymphoblastic Lymphoma/Leukemia that are clonal and transplantable to immunocompromised mice.

•  The tumors display a pre-B immunotype: B220+, CD3-, IgM-,

•  The tumor phenotype is reversible through doxycyline administration in primary and transplanted tumors (i.e. the tumors are addicted to miR-21 overexpression). •  Apoptosis is one of the major mechanisms of the tumor regression in mir-21 mice.

• A single microRNA unbalance may impact the tumor

phenotype.

• Non coding-protein genes matters!: first model of tumor

addiction to a non-coding RNA.

• miR-21 inhibition may have a therapeutic value in

lymphoma/leukemia and perhaps other cancers.

Take home message

Acknowledgments:

The Hope Funds for Cancer Research

Frank Slack Mona Nolde Xianping Liang Imran Babar Phong Trang

Animal Genomics Service Tim Nottoli Daniele Scavone Yale Mouse Research Pathology Caroline Zeiss Linda Johnson Gordon Terwilliger

Yale Cancer Center Pilot Funds

pedromedina@ugr.es pedro.medina@genyo.es http://www.ugr.es/~pedromedina

Poster Plaque Genes And Tumour Cell Proliferation In Nsclc: Prognostic Markers And Therapeutic Targets. Laura Boyero

Poster & Oral communication

SMARCA4 inactivation in lung carcinogenesis. Laura Villacorta & Ramón Santamaría.

Current work