The levels of mutant K-RAS and mutant N-RAS are rapidly reduced in a Beclin1 / ATG5 -dependent fashion by the irreversible ERBB1/2/4 inhibitor neratinib

Abstract

The FDA approved irreversible inhibitor of ERBB1/2/4, neratinib, was recently shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET and mutant K-RAS via autophagic degradation. In the present studies, in a dose-dependent fashion, neratinib reduced the expression levels of mutant K-RAS or of mutant N-RAS, which was augmented in an additive to greater than additive fashion by the HDAC inhibitors sodium valproate and AR42. Neratinib could reduce PDGFRα levels in GBM cells, that was enhanced by sodium valproate. Knock down of Beclin1 or of ATG5 prevented neratinib and neratinib combined with sodium valproate / AR42 from reducing the expression of mutant N-RAS in established PDX and fresh PDX models of ovarian cancer and melanoma, respectively. Neratinib and the drug combinations caused the co-localization of mutant RAS proteins and ERBB2 with Beclin1 and cathepsin B. The drug combination activated the AMP-dependent protein kinase that was causal in enhancing HMG Co A reductase phosphorylation. Collectively, our data reinforce the concept that the irreversible ERBB1/2/4 inhibitor neratinib has the potential for use in the treatment of tumors expressing mutant RAS proteins.

Keywords: HDAC; RAS; autophagy; neratinib; receptor tyrosine kinase.

Figure 1.

Neratinib and valproate combine to reduce the levels of K-RAS in pancreatic cancer cells. A. PANC1 cells were treated with vehicle control, neratinib (100 nM), sodium valproate (250 μM) or the drugs in combination for 24h. Cell viability was determined by trypan blue exclusion assay (n = 3 +/- SEM). B. PANC1 cells were treated with vehicle control, neratinib (100 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the expression and localization of K-RAS at 60X magnification. Arrows indicate K-RAS in vesicular structures (data from 3 separate images +/- SEM) *p < 0.05 less than vehicle control; **p < 0.05 less than value in neratinib treated cells. C. PANC1 cells were treated with vehicle control, neratinib (100 nM – 500 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the expression and localization of K-RAS at 60X magnification (data from multiple separate images & treatments +/- SEM) *p < 0.05 less than vehicle control; **p < 0.05 less than value in neratinib treated cells. D. PANC1 cells were treated with vehicle control, neratinib (100 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the expression and localization of K-RAS at 60X magnification with either ERBB1 or with cathepsin B. E. PANC1 cells were treated with vehicle control or [neratinib (100 nM) and sodium valproate (250 μM)] in combination for 6h. The cells were fixed in place and immunostaining performed at 10X to determine the expression and localization of K-RAS. Arrows indicate K-RAS in vesicular structures.

Figure 2.

Neratinib and valproate interact to reduce N-RAS levels in ovarian cancer cells and cause N-RAS to co-localize with autophagy regulatory proteins. A. Spiky ovarian cancer cells were treated with vehicle control, neratinib (100 nM), sodium valproate (250 μM) or the drugs in combination for 24h. Cell viability was determined by trypan blue exclusion assay (n = 3 +/- SEM). B. Spiky ovarian cancer cells were treated with vehicle control, neratinib (100 nM – 500 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the expression and localization of N-RAS at 60X magnification (data from multiple separate images & treatments +/- SEM) *p < 0.05 less than vehicle control; **p < 0.05 less than value in neratinib treated cells. C. Spiky ovarian cancer cells were treated with vehicle control or [neratinib (100 nM) and sodium valproate (250 μM)] in combination for 6h. The cells were fixed in place and immunostaining performed at 60X to determine the expression and localization of N-RAS. Arrows indicate N-RAS in vesicular structures. D. Spiky ovarian cancer cells were treated with vehicle control, neratinib (100 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the expression and localization of N-RAS at 60X magnification with Beclin1, LAMP2 or with cathepsin B.

Figure 3.

Neratinib and valproate down-regulate mutant N-RAS expression in PDX isolates of malignant melanoma. PDX isolates of N-RAS transformed melanoma cells were treated with vehicle control, neratinib (100 nM – 500 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the expression and localization of N-RAS at 60X magnification (data from multiple separate images & treatments +/- SEM) *p < 0.05 less than vehicle control; **p < 0.05 less than value in neratinib treated cells.

Figure 4.

Neratinib and valproate reduce the expression of N-RAS via autophagic degradation. A. Spiky ovarian cancer cells were transfected with a scrambled siRNA control or with siRNA molecules to knock down the expression of Beclin1 or ATG5. Twenty-four h after transfection, cells were treated with vehicle control, neratinib (100 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the expression and localization of N-RAS at 60X magnification. (data from multiple separate images & treatments +/- SEM) *p < 0.05 less than vehicle control; **p < 0.05 less than value in neratinib treated cells (for loading controls see Figure S6). B. and C. PANC1 and Spiky cells were treated with vehicle control, neratinib (100 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the phosphorylation of AMPKα T172 at 60X magnification. (data from multiple separate images & treatments +/- SEM) *p < 0.05 less than vehicle control; **p < 0.05 less than value in neratinib treated cells (for loading controls see Figure S6). D. Spiky cells were transfected with a scrambled siRNA control or with an siRNA to knock down the expression of AMPKα. Twenty-four hours after transfection cells were treated with vehicle control, neratinib (100 nM), sodium valproate (250 μM) or the drugs in combination for 6h. The cells were fixed in place and immunostaining performed to determine the phosphorylation of HMG CoA reductase (data from multiple separate images & treatments +/- SEM) *p < 0.05 less than vehicle control; **p < 0.05 less than value in neratinib treated cells (for loading controls see Figure S6).