GZ17-6.02 and Doxorubicin Interact to Kill Sarcoma Cells via Autophagy and Death Receptor Signaling

Abstract

GZ17-6.02 (602) is presently under phase I clinical evaluation (NCT03775525). We defined the mechanisms by which it interacted with a standard of care therapeutic doxorubicin to kill sarcoma cells. Doxorubicin and 602 interacted to rapidly activate ATM and c-MET, inactivate mTOR, AKT, and p70 S6K, enhance the expression of Beclin1 and reduce the levels of K-RAS and N-RAS. This was followed later by the drugs interacting to reduce expression of MCL-1, BCL-XL, and HDAC6. Knock down of ATM prevented the drugs alone or in combination inactivating mTOR or activating ULK1. Knock down of c-MET significantly enhanced [doxorubicin + 602] lethality. Knock down of ATM and to a greater extent ULK1, Beclin1, or ATG5 significantly reduced killing by 602 alone or when combined with doxorubicin. Expression of an activated mTOR mutant suppressed killing, autophagosome formation and prevented autophagic flux. In the absence of Beclin1, knock down of CD95, or FADD, or over-expression of c-FLIP-s or BCL-XL abolished tumor cell killing. We conclude that 602 and doxorubicin interact to increase autophagosome formation and autophagic flux as well as causing elevated death receptor signaling resulting in mitochondrial dysfunction and tumor cell death.

Keywords: CD95; GZ17-6.02; autophagy; death receptor; doxorubicin; sarcoma.

Figure 1

GZ17-6.02 and doxorubicin interact to kill sarcoma cells. (A) HT1080 and MES human sarcoma cells were treated with vehicle control, GZ17-6.02 (2 μM final curcumin), doxorubicin (200 nM) or the drugs in combination for 12–24 h. Cells were isolated, and viability determined by a trypan blue exclusion assay (n = 3 +/–SD). ^#p < 0.05 greater than either 602 or doxorubicin alone. (B) Single cells were plated into 60 mm dishes (500/dish, six separate dishes from two independent thawed cell vials). Twelve hour after plating cells were treated with vehicle control, 602 (0.5–2.0 μM), doxorubicin (0.5–2.0 μM) or the drugs in combination at a fixed dose ratio. Twenty-four hour after drug exposure, the growth media was removed, cells washed with drug free media and then cells were cultured for an additional 7 days in drug free media. Cells were fixed in place, stained with crystal violet and the number of colonies, a group of >50 cells, counted. The synergy of drug interaction was determined via the Method of Cho and Tallalay using Calcusyn for Windows program. A combination index (CI) value of <1.0 is considered synergistic.

Figure 2

GZ17-6.02 and doxorubicin interact after a 3 h exposure to activate an ATM-AMPK-ULK1- autophagy pathway concomitant with inactivation of mTOR and increased Beclin1 expression. HT1080 human sarcoma cells were treated with vehicle control, GZ17-6.02 (2 μM final curcumin), doxorubicin (200 nM) or the drugs in combination for 3 and 6 h. Cells were fixed in place and immunostained to detect the total expression and the total phosphorylation of the indicated proteins. The phosphorylation of each phosphoprotein was corrected for total protein expression; non-phosphoproteins had their expression corrected using invariant ERK2 expression. (n = 3 +/–SD) *p < 0.05 less than vehicle control; ^#p < 0.05 greater than vehicle control.

Figure 3

GZ17-6.02 and doxorubicin interact after a 6 h exposure to activate an ATM-AMPK-ULK1- autophagy pathway concomitant with inactivation of mTOR and increased Beclin1 expression. HT1080 human sarcoma cells were treated with vehicle control, GZ17-6.02 (2 μM final curcumin), doxorubicin (200 nM) or the drugs in combination for 3 and 6 h. Cells were fixed in place and immunostained to detect the total expression and the total phosphorylation of the indicated proteins. The phosphorylation of each phosphoprotein was corrected for total protein expression; non-phosphoproteins had their expression corrected using invariant ERK2 expression. (n = 3 +/–SD) *p < 0.05 less than vehicle control; ^#p < 0.05 greater than vehicle control.

Figure 4

GZ17-6.02 and doxorubicin interact to activate c-MET. HT1080 human sarcoma cells were treated with vehicle control, GZ17-6.02 (2 μM final curcumin), doxorubicin (200 nM) or the drugs in combination for 3 and 6 h. Cells were fixed in place and immunostained to detect the total expression and the total phosphorylation of the indicated proteins. The phosphorylation of each phosphoprotein was corrected for total protein expression; HDAC6 had its expression corrected using invariant ERK2 expression. (n = 3 +/–SD) *p < 0.05 less than vehicle control; ^#p < 0.05 greater than vehicle control.

Figure 5

GZ17-6.02 and doxorubicin individual and combination lethality is suppressed by knock down of ULK1, Beclin1, or ATG5. HT1080 and MES human sarcoma cells were transfected with a scrambled siRNA control (siSCR) or siRNA molecules to knock down the indicated proteins. Twenty-four hour afterwards, cells treated with vehicle control, GZ17-6.02 (2 μM final curcumin), doxorubicin (200 nM) or the drugs in combination for 12 h. Cells were isolated, and viability determined by a trypan blue exclusion assay (n = 3 +/–SD). *p < 0.05 less than vehicle control; **p < 0.05 less than corresponding value in siATM or siAMPKα transfected cells. The red, yellow and green lines are included for purposes of comparison between the individual treatments and each of the specific transfections.

Figure 6

Expression of activated mTOR suppresses tumor cell killing and abolishes autophagic flux. (A) HT1080 human sarcoma cells were transfected with an empty vector plasmid (CMV) or with a plasmid to express a mutant active mTOR protein. Twenty-four hour afterwards, cells treated with vehicle control, GZ17-6.02 (2 μM final curcumin), doxorubicin (200 nM) or the drugs in combination for 12 h. Cells were isolated, and viability determined by a trypan blue exclusion assay (n = 3 +/–SD). *p < 0.05 less than vehicle control. (B) HT1080 cells were transfected with an empty vector plasmid (CMV) or with a plasmid to express a mutant active mTOR protein and in parallel all transfected with a plasmid to express LC3-GFP-RFP. Twenty-four hour afterwards, cells treated with vehicle control, GZ17-6.02 (2 μM final curcumin), doxorubicin (200 nM) or the drugs in combination for 4 or 8 h. At each time point the mean number of intense GFP+ and RFP+ vesicles was determined counting > 40 cells per condition. (n = 3 +/–SD) ^#p < 0.05 greater than corresponding values after 4 h; ^¶p < 0.05 less than corresponding values after 4 h; ^§p < 0.05 less than corresponding values in CMV transfected cells.

Figure 7

In the absence of autophagy, GZ17-6.02 -induced killing is mediated via CD95-FADD. (A) HT1080 human sarcoma cells were transfected with a scrambled siRNA (siSCR) or with an siRNA to knock down the expression of Beclin1, and in parallel, siBeclin1 transfected cells were transfected to knock down the expression of the indicated proteins. Twenty-four hour afterwards, cells treated with vehicle control or with [GZ17-6.02 (2 μM final curcumin) + doxorubicin (200 nM)] in combination for 12 h. Cells were isolated, and viability determined by a trypan blue exclusion assay (n = 3 +/–SD). *p < 0.05 less than vehicle control; **p < 0.05 less than corresponding value in siBeclin1 alone. (B) HT1080 cells were transfected with a scrambled siRNA (siSCR) or with an siRNA to knock down the expression of Beclin1, and in parallel, cells were transfected with an empty vector plasmid or with plasmids to express BCL-XL, c-FLIP-s or dominant negative caspase 9. Twenty-four hour afterwards, cells treated with vehicle control or with [GZ17-6.02 (2 μM final curcumin) + doxorubicin (200 nM)] in combination for 12 h. Cells were isolated, and viability determined by a trypan blue exclusion assay (n = 3 +/–SD). *p < 0.05 less than vehicle control; **p < 0.05 less than corresponding value in siBeclin1 alone.

Figure 8

GZ17-6.02 regulates the expression and location of HDAC proteins. HT1080 and MES human sarcoma cells were transfected with a scrambled siRNA (siSCR) or with an siRNA to knock down the expression of Beclin1. Twenty-four hour afterwards, cells treated with vehicle control or with [GZ17-6.02 (2 μM final curcumin) for 6 h. Cells were fixed in place and immunostaining performed to detect the expression of HDACs1–11 and total ERK2 (n = 3 +/–SD). *p < 0.05 less than vehicle control; ^#p < 0.05 greater than vehicle control.

Figure 9

GZ17-6.02 regulates the expression and location of HDACs. (A,B) HT1080 cells were transfected with a scrambled siRNA (siSCR) or with an siRNA to knock down the expression of AMPKα. Twenty-four hour afterwards, cells treated with vehicle control or with [GZ17-6.02 (2 μM final curcumin) 1 h. Cells were fixed in place and immunostaining performed to detect the expression of HDACs4/5/7 and P-HDACs4/5/7 and total ERK2 (n = 3 +/–SD). *p < 0.05 less than vehicle control (n = 3 +/–SD). *p < 0.05 less than vehicle control.

Figure 10

GZ17-6.02 causes some HDACs to leave the nucleus; some to enter the nucleus and some do not move. HT1080 cells were transfected with a scrambled siRNA (siSCR) or with an siRNA to knock down the expression of AMPKα. Twenty-four hour afterwards, cells treated with vehicle control or with [GZ17-6.02 (2 μM final curcumin) 1 h. Cells were fixed in place and immunostaining performed to detect the expression and localization of HDACs1/8/9/10/11.

Figure 11

Nuclear exit of HDACs1/8/10 requires signaling by the AMPK. HT1080 cells were transfected with a scrambled siRNA (siSCR) or with an siRNA to knock down the expression of AMPKα. Twenty-four hour afterwards, cells treated with vehicle control or with [GZ17-6.02 (2 μM final curcumin)] 1 h. Cells were fixed in place and immunostaining performed to detect the expression and localization of HDACs1/8/10.