MicroRNAs (miRNA) in Gastrointestinal Cancers

Lezione di biotecnologie in Oncologia

Prof. Roberto Ria

MicroRNAs (miRNA)

• miRNA is a class of genes

encoding short RNAs (20-22 nt) discovered in 1993 able to regulate gene expression

• Present in all eukaryotes

• Phylogenetically highly conserved of the target regions in the 3’UTR

• play key roles in many biological processes, including: development cell,differentiation cell proliferation, apoptosis response to stress and oncogenesis

MMMicroRNAs (miRNA) : functions

• Produced by precursors of 90-100 nt (TU) transcript independently (also polycistronic) or derived from intronic ones

• “Seed sequence” (2-7 nt) in the

5’ UTR

MicroRNAs (miRNA)

• bind imperfectly the 3’ untranslated region of target mRNAs

miRNA: structure and biogenesis

Winter et al.,2009 – Nature Cell Biology 11, 228 - 234

•The genes coding for the microRNA (miRNA) are transcribed as long primary transcripts (pri-miRNA) •The pri-miRNA is cut from the complex formed by Drosha and DGCR8/Pasha forming the precursor miRNA (~ 70 nucleotides) •The transport of the precursors of micorRNA (premiRNA) in the cytoplasm is mediated by exportin-5 •in the cytoplasm, the complex formed by Dicer and TRBP cuts the pre-miRNA to form the duplex miRNA •microRNA is incorporated together with a Argonaute protein (AGO) RNA-induced silencing complex (RISC)

mechanisms of microRNA-mediated

repression Post-trascriptional regulation

• high sequence homology between miRNA and mRNA target • primary mechanism of regulation of microRNA plant

• imperfect complementarity between miRNA and mRNA target • primary mechanism of regulation of microRNAs of animals

Filipowicz et al. 2008, Nature Reviews genetics, 9(2):102-114

mechanisms of microRNA-mediated repression Possible mechanisms of repression of translation

Fabian et al. 2010 , Annu. Rev. Biochem. 79:351–379.

Techniques of analysis of miRNA signature

advantages: •high sensitivity and specificity requires •low quantities of material

disadvantages: • the limited size and the heterogeneous GC content of microRNAs hampers the design of the primers

1. RT-Real Time PCR

2. Microarray advantages: •high-throughput •low costs

disadvantages: • the limited size of microRNAs • possible cross-hybridization

Techniques of analysis of miRNA signature

3. Massive sequencing

Hafner et al. 2008, Methods, 44: 3–12

advantages:

• high-throughput identification of new miRNA • discrimination between miRNA similar

disadvantages:

• data analysis is more complex and presents computational requirements greater than the microarray

MicroRNAs in cancer Deletion 13q14 (miR-15a, miR16a) is the most common chromosomal aberration associated with human CLL 20% analysed miRNA were associated with fragile sites of the human genome miR-21 is overespressed in breast , colorectal , lung, pancreatic cancer, leukeamia and lymphoma

Regulation of tumorigenesis by miRNA

Yi W Kong et al Lancet Oncol 2012; 13: e249–58.

Angiogenesis, epithelial-mesenchymal transition and metastasis

Yi W Kong et al Lancet Oncol 2012; 13: e249–58.

Cancer cell invasion: activation of proteolytic enzymes, ( metalloproteases), degradation of extracellular matrix, transition of the cancer phenotype from epithelial to mesenchymal (EMT) and translocation of cancer cells.

• Angiogenesis : activation of proliferation and migration pathways in vascular smooth muscle cells

miR-143 by targeting the versican protein involved in migration induced by PDGF; downregulated in colon and

gastric cancer

The miR-200 family, miR-27, inhibit ZEB1 and ZEB2, which are

repressors of E-cadherin expression.

The Association between Two MicroRNA Variants (miR-499, miR-149) and Gastrointestinal Cancer Risk:

A Meta-Analysis

Li Li et al. 2013 , Plos one 8(11):102-114

miRNAs in Esophageal Tumors

• Esophageal cancers are classified into 2 major histopathologic subtypes: ESCC and EAC • miR-21 and miR-155 were overexpressed in ESCCs; let-7c, miR-1, miR-100,miR-143, miR-145 were underexpressed in ESCC tissues

• many miRNAs up-regulated in EAC were also up-regulated in BE tissues; Many miRNAs down-regulated in EACs were also down-regulated in BE tissues.

• Dysregulation of the miRNAs occurs along the entire continuum of progression from BE to EAC

Jee Hoon Song et al. 2012, Gastroenterology 2012;143:35–47

miRNAs in Gastric Cancer 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-221-222, 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

abnormal methylation patterns lead to gastric carcinogenesis through the hypermethylated promoter of tumor suppressor genes

miRNAs in Gastric Cancer

• MicroRNA-148a is

downregulated in gastric cancer

• MMP7 is a direct and functional target of miR-148a

• hypermethylation of MIR148A CpG islands is correlated with suppression of miR-148a in GC cell lines

• miRNA 148a indicates tumor invasiveness and poor prognosis

Sakamoto et al. 2014, Cancer Sci, 104 (2) 236-243

miRNAs in Gastric Cancer

•PTEN gene is an important regulator of protein phosphatases and 3‘ phosphoinositol phosphatases •PTEN is inactivated in some malignant tumors, resulting in Akt hyper-activation, there by promoting cell proliferation, inhibition of apoptosis •It is a target gene of the miR-221 and miR-222 cluster •radiotherapy as a standard treatment for patients with a high risk of recurrence •increasing PTEN expression by silencing miR-221/222 could enhance the radiosensitivity of tumor cells

microRNA-221 and microRNA-222 up regulate in gastric cancer

Chun-zhi et al. BMC Cancer 2010, 10:367

miRNAs in HCC

miR-221

DOWN is a key regulator of the differentiation of adult hepatocytes via repression of genes not specific to the liver

miR-122

Chun-zhi et al. BMC Cancer 2010, 10:367

UP miR-221 is a paradigmatic example of an miRNA regulating multiple pathways (CDKN1B/p27, CDKN1C/p57, BH3, PTEN, mTOR)

miRNAs in HCC

miR-199

DOWN leads to cell cycle arrest at G1 phase, reduces invasive capability, and enhances susceptibility to hypoxia. These effects could be explained by modulation of target genes, such as MET, mTOR, and HIF-1α.

miR-21

UP Overexpression of miR-21 can protect against apoptosis and increase tumor cell proliferation and migration. The most important of target is PTEN, which promotes cell survival via activation of the PI3K-AKT pathway Chun-zhi et al. BMC Cancer 2010, 10:367

miRNAs in colon and rettal cancer miR-21 functions in many cell types as an anti apoptotic and pro-survival factor

increased expression of several miRNAs such as miR-21, miR-31, miR-96, miR-221, has been shown to correlate with the presence of adenomas

Yamamichi et al analysed miR-21 expression patterns in different stages of CRC development. the frequency and extent of miR-21 expression increased during the transition from precancerous CRA to advanced carcinoma.

expression of miR21 in benign colon adenomas may represent an early event in the progression to carcinoma

microRNA in metastatic colorectal cancer

miR-103/107 + hypoxia

promotes tumor metastasis through

trans-activating metastasis-

inducing HGF/MET signaling pathway

DAPK e KLF4

inactivate integrin β1

miR-17/92 PTEN e BCL2-L11 Angiogenesis

miR-143

Tokarz et al. ABP 2012, 10:367

MACC1

HGF / MET

promote cell–matrix interaction and downregulate

E-cadherin/claudin-3/occludin to

diminish cell–cell adhesion, increase

cell motility.

miRNA in pancreatic cancer Comparing differential expression of blood-based circulating miRNAs between early stage pancreatic cancer patients (n = 8) and healthy controls (n = 11).

mirR 642B-3p , miR-885-5p e miR-22-3p up regulated in cancer patients can differentiate pancreatic cancer patients from healthy controls with high sensitivity and specificity (91%)

Ganepola et al. WJGO 2014, 10:367

MiR-885-5p activates the p53 pathway, causes down-regulation of CDK and mini-chromosome maintenance protein,and suppresses matrix metallopeptidase 9 expression and Caspase genes

miR-22-3p inhibits cell cycle progression by repressing Max and ErbB3 expression post-transcriptionally, mediates the effects of p53, and suppresses interferon gene expression by blocking interferon regulatory factor-5

Biomarkers in body fluids • 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

• Human plasma contains roughly 308 μg/L of RNA, in which about 350 different miRNA sequences can be detected

• miRNAs are stable in blood and exist as free miRNAs, exosomal miRNAs, or most predominantly as Argonaute2 (Ago2) protein-bound miRNAs

• Several studies have analyzed the diagnostic and prognostic value of circulating miRNAs

• Limitations of using circulating miRNAs as diagnostic markers include:

low abundance in blood

difficulties in measuring quality and quantity

normalization issues

unclear mechanisms of release and origin

Diagnosis and classification

histological examination

miRNAs SIGNATURE

limited by availability of adequately preserved tissue and the possibility

of subjective interpretation by pathologists

resistance to degradation; miRNA expression levels can be

established in a few hours with as little as 10 ng of total RNA

versus

can be used to differentiate between malignant and benign conditions in several

organs

can also be used to identify well characterised genotypes—eg, to distinguish between sporadic and germ-line tumours, or to identify genomic instability within a tumour

new studies are identifying extra cellular miRNA patterns that enable the detection of early-stage cancers

Lancet Oncol 2012; 13: e249–58.

Diagnosis in Gastrointestinal Cancers GE Cancers: • Zhang et al identified a panel of 7 serum miRNAs (miR-10a, miR-22, miR-100, miR-

127-3p, miR-133a, miR-148b, and miR-223) that was associated with ESCC

• A microarray analysis of serum miRNAs reported increased levels of miR-187, miR-

371-5p, and miR-378 in patients with advanced- stage gastric cancer compared with healthy individuals

there are still insufficient data available on the levels of circulating miRNAs in serum or plasma as a diagnostic for gastroesophageal cancers.

HCC: • miR-200c,miR-141 and miR126, could be used to distinguish primary HCC versus other tumor metastases

• miR-122 and miR-21 levels circulating have been reported by more than one study to be significantly higher in patients with HCC.

• miR-205/miR-194 expression could be used to distinguish between GI tumors and metastases outside the GI system

Diagnosis in Gastrointestinal Cancers

• miR-21 , miR-106 and miR-144 are upregulated in the faeces of patients with CRC.

Colorectal Cancer:

• mi-21, 17-3p, and 92 are elevated in patients with CRC ; the plasma levels of these markers are reduced after surgery in patients with CRC suggesting that the high levels specifically indicate the presence of a CRC

Pancreatic Cancer: The panel (mirR 642B-3p , miR-885-5p e miR-22-3p) may work

alone or in conjunction with other known immunoassays, such as CA19-9 and CEA as a diagnostic test for early stage

pancreatic cancer.

Prognostic indicators in GI cancers

High expression of miR-221 is associeted with a shorter time to recurrence in HCC

GE Cancers:

HCC:

A miRNA signature distinguishing gastric cancer from normal stomach epithelium was identified. 30 miRNAs were significantly inversely correlated with TTP whereas 28 miRNAs were significantly positively correlated with TTP of 82 cancer patients (P<0.05)

Prognostic indicators in GI cancers Colorectal Cancer:

miR-378 significantly down-regulated in CRC

vimentin as the functional downstream target of miR-378 by directly targeting the 3′-UTR

Patients with low miR-378 expression had significantly poorer OS

In vivo: After 5 weeks, miR-378 over-expressing tumors were significantly smaller than those of mice transfected with scramble control

Vimentin expression and perturbation of E-cadherin-mediated cell adhesion are therefore both regarded as hallmarks of EMT

Zhang et al. BMC Cancer 2014, 14:109

Anti-cancer anti-miR terapy? Widespread disruption of miRNAs is caused by at least three different mechanisms: • the loss, amplification or mutation of a fragile cancer-related genomic region; •the change of epigenetic control; • the abnormality of miRNA-processing steps

Treatment can be targeted to miRNAs in two ways (A and B): • miRNA reduction • miRNA replacement

Yi W Kong et al Lancet Oncol 2012; 13: e249–58.

1. Blocking oncogenic miRNAs using antisense oligonucleotides

2. Constructs Locked nucleic acid (LNA) 3. miRNA sponges 4. miR-mask 5. Small molecule inhibitors 6. Restoring the expression of tumor suppressor miRNAs 7. Reprogram cancer cells

Strategies for anti-cancer therapies based

on miRNA:

In HCC: Krutzfeldt et al. First demonstrated miR-122 knockdown in vivo by the systemic delivery of an antagomirs, which is a 2′-O-methyl-, phosphorothioate-modified, and cholesterol conjugated oligoribonucleotides complementary to specific microRNAs

The first miRNA-targeted drug, miravirsen

LNA= Locked nucleic acid

antimiR-122

The first miRNA-targeted drug, miravirsen

Clinical and pre-clinical trials in progress MicroRNA-126 and epidermal growth factor-like domain 7 – an angiogenic couple

of importance in metastatic colorectal cancer. Results from the Nordic ACT trial

The results validate the previous findings on the prognostic value of miRNA-126 in mCRC may suggest a relationship between treatment efficacy and EGFL7 expression. As miRNA-126 may target VEGF-A as well as EGFL7, the results may provide predictive information in relation to

next-generation anti-angiogenetics

T F Hansen et al British Journal of Cancer (2013) 109

Circulating miRNAs

Lezione di biotecnologie in Oncologia

Prof. Roberto Ria

Analisi dei miRNA circolanti

come biomarkers precoci di

varie patologie

I miRNAs si sono rivelati

estremamente stabili nei fluidi

biologici

Il loro profilo di espressione

spesso correla con uno

specifico stato patologico

miRNA circolanti

Carriers dei miRNAs

Uptake dei miRNAs extracellulari da parte della cellula target

Endocitosi, fagocitosi e

fusione diretta con la

membrana rappresentano

i meccanismi di ingresso

dei miRNAs associati a

vescicole (esosomi e

‘shedding vescicles’)

L’ingresso recettore-

mediato è riservato ai

miRNA associati a

proteine. Es. HDL-miRNAs

si servono del recettore

SR-BI.

Biopsia liquida

Circulating microRNAs associated with cancer

Good candidates for non-invasive diagnostic markers for cancer

Circulating miRNAs as biomarkers for hepatocellular carcinoma (HCC) diagnosis