• Debrecen, U. of (2019)

  • Cancer Biology

  • Christopher Maher, Ph.D.

  • Characterization of DDX54 functions in breast cancers.

  • h.mai@wustl.edu

Research

ADAR1 is an RNA editing enzyme which can convert adenosine to inosine in double-strand RNAs (dsRNAs) through its deaminase activities, and prevent the sensing of self dsRNAs by immune system. ADAR1 was found to be overexpressed in several types of cancers and knocking out of ADAR1 resulted in apoptosis and proliferation arrest in TNBC. By using proximity binding assay, ADAR1 was found to highly associate with DDX54 in non-TNBC and this was later confirmed by co-immunoprecipitation assay. Knocking out of DDX54 significantly reduced cell survival and proliferation in non-TNBC cell lines. Combination knocking out of DDX54 and ADAR1 further enhances the killing activities toward these cell lines. These observations suggest DDX54 as a new potential target to eliminate tumor cells. However, we have not known whether DDX54 activity is unique for non-TNBC or it is universally important for other tumor types. On the other hand, it is important to understand the mechanisms of how knocking out of DDX54 enhances apoptosis and reduce tumor proliferation. One of the hypotheses is that DDX54-knocked out cells accumulate R-loop structures which were normally cleaved by DDX54. This accumulation leads to DNA damage and genomic instability. We, therefore, hypothesize that the survival of DDX54-knocked out non-TNBC cell lines can be rescued by inducing expressions of RNAseH1, the enzyme that can cleave R-loop structures. To test these hypotheses, we will perform the following experiments:


Characterizing the effect of DDX54 loss in TNBC cell lines (MDA-MB-231):


1. We will knock out DDX54 in MDA-MB-231 cells by using lentiviral transfection of siRNA that specifically targets DDX54.


2. We will characterize the survival of MDA-MB-231 following DDX54 knocking out by using foci formation assay.


3. We will characterize other molecular responses of DDX54 knocked-out MDA-MB-231 by determining the level of PKR and dsRNA.


Rescuing the survival of DDX54 and ADAR1 double knocked out non-TNBC cells (SK-BR-3) by inducing RNase H1 expression:


1. We will clone the plasmid contains sequences that encode for lentiviruses that can induce RNase H1 expression in cell lines.


2. We will transfect SK-BR-3 (DDX54 and ADAR1 knocked-out) with the lentiviruses to induce RNAsH1 expression.


3. We will determine whether the induction of RNAseH1 expression rescue the survival of these cells by using foci formation assay.

Last Updated: 1/4/2022 12:23:17 PM

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