GZ17-6.02 and axitinib interact to kill renal carcinoma cells

Abstract

GZ17-6.02 is undergoing clinical evaluation in solid tumors and lymphoma. The present studies were performed to define its biology in renal carcinoma cells and to determine whether it interacted with axitinib to enhance tumor cell killing. GZ17-6.02 interacted in an arithmetically greater than additive fashion with axitinib to kill kidney cancer cells. GZ17-6.02 and axitinib cooperated to inactivate ERBB2, c-MET, c-KIT, c-SRC, the AMPK, STAT3, STAT5 and eIF2α and to activate PERK, ULK1 and ATG13. The drugs interacted to increase the expression of FAS-L and to decrease the levels of MCL1, BCL-XL, and HDACs 1-3. The drugs as single agents inactivated the Hippo pathway. GZ17-6.02 and axitinib interacted to enhance autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5, eIF2α, toxic BH3 domain proteins or CD95/FADD significantly reduced drug combination lethality. GZ17-6.02 and axitinib increased the expression of BAK, BIM, Beclin1 and ATG5, effects blocked by knock down of eIF2α. The drugs increased phosphorylation of ULK1 S757 and ATG13 S318 and decreased the phosphorylation of mTORC1 and mTORC2, effects blocked by knock down of AMPKα. Knock down of Beclin1 or ATG5 prevented the drug combination reducing expression of HDACs 1-3 and from enhancing the expression of MHCA. Knock down of HDACs 1-3 enhanced MHCA expression. We conclude that GZ17-6.02 and axitinib interact to kill requiring ER stress signaling, autophagy and death receptor signaling. Autophagic degradation of HDACs played a key role in enhancing MHCA expression and of a potential improved response to checkpoint inhibitory immunotherapy.

Keywords: GZ17-6.02; HDAC; autophagy; axitinib; renal cell carcinoma.

Figure 1. GZ17-6.02 and axitinib regulate protein expression and protein phosphorylation in UOK121LN RCCs.

(A) UOK121LN cells were treated with vehicle control, GZ17-6.02 (2 μM), axitinib (50 nM) or the drugs in combination for 24 h. Cells were isolated, and viability determined by trypan blue exclusion. (n = 3 +/− SD). (B) UOK121LN cells were treated with vehicle control, GZ17-6.02 (2 μM), axitinib (50 nM) or the drugs in combination for 6 h. Cells were fixed in place and in-cell immunostaining performed to determine alterations in protein expression and protein phosphorylation with ERK2 as an invariant loading control (n = 3 +/− SD). ^* p < 0.05 less than vehicle control; ^# p < 0.05 greater than vehicle control.

Figure 2. GZ17-6.02 and axitinib regulate protein expression and protein phosphorylation in A498 RCCs.

(A) A498 cells were treated with vehicle control, GZ17-6.02 (2 μM), axitinib (50 nM) or the drugs in combination for 24 h. Cells were isolated, and viability determined by trypan blue exclusion. (n = 3 +/− SD). (B) A498 cells were treated with vehicle control, GZ17-6.02 (2 μM), axitinib (50 nM) or the drugs in combination for 6 h. Cells were fixed in place and in-cell immunostaining performed to determine alterations in protein expression and protein phosphorylation with ERK2 as an invariant loading control (n = 3 +/− SD). ^* p < 0.05 less than vehicle control; ^# p < 0.05 greater than vehicle control.

Figure 3. GZ17-6.02 and axitinib interact to cause toxic autophagosome formation and autophagic flux.

(A) and (B) A498 and UOK121LN cells were transfected with a plasmid to express LC3-GFP-RFP. After 24 h, cells were treated with vehicle control, GZ17-6.02 (2 μM), axitinib (50 nM) or the in combination for 4 h and 8 h. The mean number of intense GFP+RFP+ and RFP+ punctae per cell was determined at each time point (n = 3 +/− SD) ^# p < 0.05 greater than corresponding value at the 4 h timepoint. (C) and (D) A498 and UOK121LN cells were transfected with a scrambled siRNA or with siRNA molecules to knock down expression of the indicated proteins. After 24 h, cells were treated with vehicle control or [GZ17-6.02 (2 μM) plus axitinib (50 nM)] in combination for 24 h. Cells were isolated, and viability determined by trypan blue exclusion. (n = 3 +/− SD). ^* p < 0.05 less than corresponding value in siSCR cells; ^‡ p < 0.05 greater than corresponding siATM and siAMPK values.

Figure 4. [GZ17-6.02 + axitinib] kill RCCs via death receptor signaling and mitochondrial dysfunction; the majority of killing downstream is caspase-independent.

(A) and (B) A498 and UOK121LN cells were transfected with a scrambled siRNA or with siRNA molecules to knock down expression of the indicated proteins. After 24 h, cells were treated with vehicle control or [GZ17-6.02 (2 μM) plus axitinib (50 nM)] in combination for 24 h. Cells were isolated, and viability determined by trypan blue exclusion. (n = 3 +/− SD). ^* p < 0.05 less than corresponding value in siSCR cells. (C) and (D) A498 and UOK121LN cells were transfected with an empty vector plasmid (CMV) or with plasmids to express BCL-XL, FLIP-s, dominant negative caspase 9, activated MEK1, activated AKT, activated mTOR or activated STAT3. After 24 h, cells were treated with vehicle control or [GZ17-6.02 (2 μM) plus axitinib (50 nM)] in combination for 24 h. Cells were isolated, and viability determined by trypan blue exclusion. (n = 3 +/− SD). ^* p < 0.05 less than corresponding value in CMV cells; ^‡ p < 0.05 greater than corresponding values in cells transfected to express BCL-XL or FLIP-s; ^† p < 0.05 less than corresponding values in cells transfected to express activated MEK1, activated AKT or activated mTOR.

Figure 5. Knock down of ATM prevents [GZ17-6.02 + axitinib] regulating the phosphorylation of mTOR, ULK1 and ATG13.

A498 and UOK121LN cells were transfected with a control siRNA or with an siRNA to knock down the expression of ATM. After 24 h, cells were treated with vehicle control or [GZ17-6.02 (2 μM) plus axitinib (50 nM)] in combination for 6 h. Cells were fixed in place and the expression and phosphorylation of ULK1, ATG13, mTORC1, mTORC2 and ERK2 determined (n = 3 +/− SD) ^* p < less than corresponding value in siSCR cells; ^# p < greater than corresponding value in siSCR cells.

Figure 6. Knock down of AMPKα prevents [GZ17-6.02 + axitinib] regulating the phosphorylation of mTOR, ULK1 and ATG13.

A498 and UOK121LN cells were transfected with a control siRNA or with an siRNA to knock down the expression of AMPKα. After 24 h, cells were treated with vehicle control or [GZ17-6.02 (2 μM) plus axitinib (50 nM)] in combination for 6 h. Cells were fixed in place and the expression and phosphorylation of ULK1, ATG13, mTORC1, mTORC2 and ERK2 determined (n = 3 +/− SD) ^* p < less than corresponding value in siSCR cells; ^# p < greater than corresponding value in siSCR cells.

Figure 7. Knock down of eIF2α prevents [GZ17-6.02 + axitinib] increasing the expression of BAK, BIM, Beclin1 and ATG5.

A498 and UOK121LN cells were transfected with a control siRNA or with an siRNA to knock down the expression of eIF2α. After 24 h, cells were treated with vehicle control or [GZ17-6.02 (2 μM) plus axitinib (50 nM)] in combination for 6 h. Cells were fixed in place and the expression of BAK, BIM, Beclin1, ATG5 and ERK2 determined (n = 3 +/− SD) ^* p < less than corresponding values in siSCR cells.

Figure 8. Knock down of [HDAC2 + HDAC3] or [HDAC1 + HDAC3] regulates MHCA expression in RCCs.

(A) A498 and UOK121LN cells were transfected with a scrambled siRNA control or with siRNA molecules to knock down the expression of Beclin1 or ATG5. After 24 h, cells were treated with vehicle control or [GZ17-6.02 (2 μM) plus axitinib (50 nM)] in combination for 6 h. Cells were fixed in place and the expression of HDAC1, HDAC2, HDAC3, MHCA and ERK2 determined (n = 3 +/− SD) ^* p < less than corresponding values in siSCR cells. (B) A498 and UOK121LN cells were transfected with a scrambled siRNA control or with siRNA molecules to knock down the expression of HDAC1, HDAC2 and HDAC3, as indicated. After 24 h, cells were fixed in place and the expression of MHCA and ERK2 determined (n = 3 +/− SD) ^# p < 0.05 greater than siSCR control.

Figure 9. GZ17-6.02 regulates MHCA expression in a variety of tumor cell types and is more capable of regulating MHCA levels than HDAC inhibitors.

Tumor cells (renal: UOK121LN, A498; breast: BT483, MCF7; hepatoma: HEPG2, HEP3B; colorectal: HCT116, HT29) were treated with vehicle control or with GZ17-6.02 (2 μM), sodium valproate (250 μM), vorinostat (250 nM) or entinostat (50 nM) for 6 h. The expression of MHCA and ERK2 determined (n = 3 +/− SD) ^# p < 0.05 greater than siSCR control.

Figure 10. Control siRNA knock down and protein over-expression in RCCs.

Cells were transfected to over-express or knock down protein expression. Twenty-four h after transfection, cells were fixed in place and immuno-stained to determine protein levels with invariant ERK2 as a loading control (n = 3 +/− SD).