Pizza Club

Matthieu GOBIN 15/03/2016

Glioblastoma (GBM): treatment strategy

- Most deadly brain tumour with a mean survival of 15 months - Standard therapy: IR + concomitant TMZ (alkylating agent)

-> induction of DNA lesions (Me-, Double stranded breaks) in order to cause cell death

- DNA repair has an important role in the resistance of the tumour

- Critical step is to find biomarkers and novel targets to better tailor the patient’s treatment

-> target the radioresistance of GBM to potentiate current treatment regimen

microRNAs (miRs): Cellular function

-> small non-coding RNAs (20-22 nt)

-> inhibition of mRNA translation (mRNA degradation or repression)

-> regulatory mechanism

-> RNA interfering technology (shRNA, siRNA)

Figure1. miRNA screen for radioresistance

A: miRs screen workflow (275 miRs)

B: Identification of 40 miRs IR-independant and 4 miRs IR-dependant-> conferred a growth advantage-> miR-1 / 125a / 150 / 425 will be used for functional studies

C: Measure of radiosensitivity by individual expression of 4 miRs upon IR treatment-> correlation of results obtained in B

Figure 2. Radioresistance and miRNA levels in GBM cell lines

A: Assessment of GBM cell line specific radiosensitivity

-> LN229 = most sensitive-> U251 = most resistant

B: Expression of 4 miRs in GBM cell lines-> LN229 = low expression-> U251 = high expression

Assessment of radioresistance and miRs expression in GBM cell lines-> Selection of LN229 and U251 as appropriate candidate cell lines for further testing

Figure 3. miR-1, miR-125a, miR-150 and miR425 regulate radioresistance

A: Individual expression of miRs induces radioresistanceB: Tandem expression promotes radioresistance

-> suggesting that the 4 miRs can work in isolation or together to promote

radioresistance

Radiosensitive GBM cell line

C: Individual inhibition of miRs induces radiosensitivityD: Expression of miR sponges promotes radiosensitivity

-> suggesting that the 4 miRs may be important targets for prevention of therapeutic resistance

Radioresistant GBM cell line

Figure 4. Impact of miRNAs on DNA damage response

A-C: Tandem miRs expression decreases IR induced apoptosis

-> expression and activity of major apoptosis factors is decreased (caspases)

D-I: miRs expression induces phosporylation of the major DNA damage checkpoint factors

-> activation of cell cycle checkpoint kinases

-> enhancement of DNA lesions due to miRs expression?

J+K: H2AX activation is not altered upon miRs expression

-> DNA repair is not altered upon overexpression of these 4 specific miRs

Figure 5. Identification of the regulatory pathways of miRNAs

A: In silico based prediction of TF binding to the promoters of the 4 selected miRs

-> TGF-β most significant TF

B: TGF-B induces expression of a specific miRs

-> miR-1 in NHA and miR 125a in U87-> TGF-β might induce radioresistance?

C+D: Specific miR inhibition neutralizes radioresistance caused by TGF-B signaling

-> TGF-β induces radioresistance

E: TCGA probing of TGF-B signaling and miR-1/125a expression

-> statistical significant in this primary GBM cohort

F: Expression of the 4 miRs in humand GBMs and specifically in primary GBMs

-> the 4 identified miRs are potentially important for this cancer

Summary of results

- Identification of 4 miRs conferring radioresistance-> miR-1 / 125a / 250 / 425

- Functional analysis shows that they seem to limit apoptosis induced by IR and have no effect on DNA repair efficiency

- miR-1 and 125a seem to be expressed in response to TGF-β signaling

- TGF-β alone is able to reverse the redaiosensitive effect of miR-1 and 125a inhibition

- Expression of these 4 miRs in primary GBMs samples form the TCGA cohort

1) Targeting of these miRs could be of potential benefit for the patients

2) Assessment of the expression of these miRs in cancer could pinpoint their radiosensitivity (better tailor the treatment strategy)

Discussion

- Use of non-GBM cell line (U2OS) to identify IR-dependant miRs-> why not test the miRs in a GBM cell line context?

- Assessment of the impact of miRs expression on DNA repair efficiency-> only use checkpoint kinases which are early events and could be activated through other mechanisms during IR

- TGF-β signaling known to mediate response to radiation and this pathway is rather broad and can be activated very easily

- Lack of pre-clinical validation experiments and use of more biologically relevant GBM spheroid cell lines

- Still major limitations nowadays with the prediction of miRs targets -> try to tackle this question by using RNA22 algorithm and probing predicted targets from a genome wide RNAi-screen -> significant overlap with their results (DNA repair, cell cycle, apoptosis)