Knockdown of MAGEA6 Activates AMP-Activated Protein Kinase (AMPK) Signaling to Inhibit Human Renal Cell Carcinoma Cells

Background/Aims: Melanoma antigen A6 (MAGEA6) is a cancer-specific ubiquitin ligase of AMP-activated protein kinase (AMPK). The current study tested MAGEA6 expression and potential function in renal cell carcinoma (RCC). Methods: MAGEA6 and AMPK expression in human RCC tissues and RCC cells were tested by Western blotting assay and qRT-PCR assay. shRNA method was applied to knockdown MAGEA6 in human RCC cells. Cell survival and proliferation were tested by MTT assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by the TUNEL assay and single strand DNA ELISA assay. The 786-O xenograft in nude mouse model was established to test RCC cell growth in vivo. Results: MAGEA6 is specifically expressed in RCC tissues as well as in the established (786-O and A498) and primary human RCC cells. MAGEA6 expression is correlated with AMPKα1 downregulation in RCC tissues and cells. It is not detected in normal renal tissues nor in the HK-2 renal epithelial cells. MAGEA6 knockdown by targeted-shRNA induced AMPK stabilization and activation, which led to mTOR complex 1 (mTORC1) in-activation and RCC cell death/apoptosis. AMPK inhibition, by AMPKα1 shRNA or the dominant negative AMPKα1 (T172A), almost reversed MAGEA6 knockdown-induced RCC cell apoptosis. Conversely, expression of the constitutive-active AMPKα1 (T172D) mimicked the actions by MAGEA6 shRNA. In vivo, MAGEA6 shRNA-bearing 786-O tumors grew significantly slower in nude mice than the control tumors. AMPKα1 stabilization and activation as well as mTORC1 in-activation were detected in MAGEA6 shRNA tumor tissues. Conclusion: MAGEA6 knockdown inhibits human RCC cells via activating AMPK signaling.

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Bromodomain-Containing Protein 4 (BRD4) Inhibition Sensitizes Palomid 529-Induced Anti-Renal Cell Carcinoma Cell Activity in Vitro and in Vivo

Cellular Physiology and Biochemistry ◽

10.1159/000494185 ◽

2018 ◽

Vol 50 (2) ◽

pp. 640-653 ◽

Cited By ~ 2

Author(s):

Zhao-yu Xing ◽

Yin Wang ◽

Long Cheng ◽

Jie Chen ◽

Xiao-zhou He ◽

...

Keyword(s):

Renal Cell Carcinoma ◽

Cell Growth ◽

Cell Carcinoma ◽

Cell Apoptosis ◽

Renal Cell ◽

Elisa Assay ◽

Dual Inhibitor ◽

Brd4 Inhibition

Background/Aims: Mammalian target of rapamycin (mTOR) is a valuable treatment target of renal cell carcinoma (RCC). Palomid 529 is a novel mTORC1/2 dual inhibitor. Methods: RCC cells were treated with different concentrations of Palomid 529. Cell survival was tested by MTT assay and clonogenicity assay. Cell proliferation was tested by BrdU ELISA assay. Cell apoptosis was tested by the Hoechst-33342 nuclei staining assay and Histone DNA ELISA assay. mTOR signaling was tested by Western blotting assay and co-immunoprecipitation (IP) assay. The SCID mouse 786-O xenograft model was established to test RCC cell growth in vivo. Results: Palomid 529 exerted cytotoxic, anti-proliferative and pro-apoptotic activities in 786-O RCC cells. Palomid 529 disassembled mTORC1/2, causing de-phosphorylation of mTORC1/2 substrates. Bromodomain-containing protein 4 (BRD4) is a primary resistant factor of Palomid 529. Palomid 529-induced 786-O cell apoptosis was sensitized by BRD4 inhibitors or BRD4 silencing, but inhibited with BRD4 over-expression. Palomid 529-induced cytotoxicity in the primary human RCC cells was negatively correlated with BRD4 expression level. In vivo, Palomid 529 i.p. administration inhibited 786-O xenograft tumor growth in SCID mice. Its anti-tumor activity was further sensitized by co-administration of the BRD4 inhibitor JQ1. Cconclusion: Palomid 529 inhibits RCC cell growth in vitro and in vivo. BRD4 inhibition could further sensitize Palomid 529 against RCC cells.

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