PF575 OVEREXPRESSION DNP73 PROMOTES THE PROLIFERATION AND DRUG RESISTANCE OF MULTIPLE MYELOMA
Authors: Xu J. , Liu L. , Du C. , Yu Z. , Wei X. , Qiu L. , Hao M. 
Published: June 2019

Background:

Genomic instability and chromosomal abnormalities induced by DNA damage are important biological characteristics of multiple myeloma (MM). DNp73 is one of the main isomers of TP73, a member of the TP53 protein family. We found that DNp73 was highly expressed in MM which could resist the apoptosis induced by DNA damage in MM cells, leading to clinical drug resistance.

Aims:

In order to further explore the mechanism of DNp73 mediating drug resistance to DNA damage in MM, this study verified and explored from multiple levels including cell phenotype changes in vivo and in vitro.

Methods:

MM cell lines OCI‐MY5 and ARP1 were used to constructed DNp73 overexpression (OE) cell lines, respectively. Cell proliferation activity of DNp73 OE and EV (empty vector) cell lines was compared by absolute cell count method and CCK8 method, and cell proliferation was determined under the action of doxorubicin (5nM &50 nM) or epirubicin (5nM & 10 nM) at different concentrations. The cloning formation and drug sensitivity of ARP1 DNp73 OE/EV cells were determined by soft agar cloning formation assay. Flow cytometry was used to detect the apoptosis and cell cycle arrest of cells in DNp73 OE and EV cells under UV and at different concentrations of reagents treatment. Xenografted model and 5TGM1 spontaneous mouse MM model were used to detect the effect of DNp73 on the proliferation of MM cells and the treatment of DNA damage reagents in vivo, as well as the survival period of tumor‐bearing mice were analyzed. RNA‐seq and gene enrichment analysis (GSEA) were performed to explore the potential signaling pathway of DNp73 against apoptosis. Further western blotting and quantity‐real time PCR were performed for validation. ChIP and CoIP were used to search for the potential downstream key target genes and target proteins of DNp73.

Results:

DNp73‐OE ARP1 & OCI‐MY5 cell lines showed stronger proliferation activity than EV MM cell lines, and showed significant resistance to DNA damage reagents, doxorubicin and epirubicin treatment. The cloning formation experiment of soft agar confirmed that DNp73 induced the resistance of MM cells to those drugs. Flow cytometry analysis showed that DNp73 OE suppressed the MM cells apoptosis and suppressed G1 phase arrest induced by those drugs treatment (P < 0.05). In vivo experiments showed that the tumor burden of mice was significantly more severe compared with the mice of control group. The tumor‐bearing mice in DNp73 OE group were resistant to the treatment of DNA damage reagents. Moreover, the survival of DNp73 OE tumor‐bearing mice was significantly shortened compared with the control group. RNA‐seq, GSEA analysis and western blot assay revealed that DNp73 overexpression induce drug resistance and proliferation through the activation of c‐myc and AKT‐related pathways and inhibition of p21/p27 signal pathway which caused cell cycle arrested in MM.

Summary/Conclusion:

DNp73 promote MM cell proliferation, anti‐apoptosis and inhibit cell cycle arrest through activating c‐myc and Akt‐related pathways. DNp73 overexpression is related to the occurrence of drug resistance in MM.

image