MicroRNAs (miRNA) in Gastrointestinal Cancers

UNIVERSITA’ DEGLI STUDI DI BARI “ALDO MORO” DIPARTIMENTO DI SCIENZE BIOMEDICHE ED ONCOLOGIA UMANA

U.O.C. ONCOLOGIA MEDICA UNIVERSITARIA SCUOLA DI SPECIALIZZAZIONE IN ONCOLOGIA MEDICA

Direttore: Prof. Franco Silvestris

MicroRNAs (miRNA) [1]

• miRNA is a class of genes

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

• Present in all eukaryotes

• Target regions in the 3’UTR are Phylogenetically highly conserved

• Play key roles in many biological processes, including: cell growth, differentiation, proliferation, apoptosis stress response and oncogenesis

• Produced by precursors (also polycistronic) of 90-100 nt (TU) which are independently transcribed, or derived from intronic TU

• “Seed sequence” (2-7 nt) in the

5’ UTR

MicroRNAs (miRNA) [2]

• bind imperfectly the 3’ untranslated region of mRNA targets

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 miRNA precursor (~ 70 nucleotides) •The transport of the precursors of microRNA (pre-miRNA) 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 in plants

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

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

Mechanisms of microRNA-mediated repression Possible mechanisms of translation repression

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

Techniques of analysis of the miRNA signature [1]

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

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

1. Real Time PCR

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

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

Techniques of analysis of miRNA signature [2]

3. Massive sequencing

Hafner et al. 2008, Methods, 44: 3–12

advantages:

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

disadvantages:

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

MicroRNAs in cancer

Deletion 13q14 is the most common chromosomal aberration associated with human CLL (miR 15 a-miR16 a) 20% analysed miRNA were associated with fragile sites of the human genome miR-21 is overespressed in breast, colorectal, lung, and 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 targets the versican protein involved in migration induced by PDGF;

and it is downregulated in colon and gastric cancers

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

repressors of the 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 miRNA 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 [1]

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 [2]

• 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 [3]

•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, thereby promoting cell proliferation and 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 can enhance the radiosensitivity of tumor cells

microRNA-221 and microRNA-222 are upregulated in gastric cancer

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

miRNAs in HCC [1]

miR-221

DOWN is a key regulator of the differentiation of adult hepatocytes via repression of specific liver genes

miR-122

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

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

miRNAs in HCC [2]

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 the 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 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 colorectal 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 increase 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 interactions and

downregulate E-cadherin/claudin-

3/occludin to diminish cell–cell

adhesion and increase cell motility.

miRNA in pancreatic cancers

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 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 their 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

only 10 ng of total RNA

versus

can be used to differentiate malignant from benign conditions in several organs

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

new studies are identifying extracellular miRNA patterns which enable the detection of early-stage cancers

Lancet Oncol 2012; 13: e249–58

Diagnosis in Gastrointestinal Cancers [1]

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 (sensitivity 87% and specificity 70%)

there are still insufficient data available on the levels of circulating miRNAs in serum or plasma as a diagnostic biomarker 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 circulating levels have been reported in many studies 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 [2]

• 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 their 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 [3]

High expression of miR-221 is associated 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 [4] Colorectal Cancer:

miR-378 is significantly down-regulated in CRC

Vimentin as the functional downstream target of miR-378

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 alteration 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 therapy? 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

• 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:

HCC: Krutzfeldt et al. 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. With LNA modification (2′-O-methyl modification) the length of antimir was shortened significantly to cover only the seed sequence of selected miRNAs, resulting in much higher affinity compared to the fulllength antimirs . LNA-modified antimirs allows low off-target effects, efficient suppression of entire families of miRNA, and broad distribution throughout different organs after systemic delivery

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

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