Neratinib augments the lethality of [regorafenib + sildenafil]

Abstract

Regorafenib is approved for the treatment of colorectal cancer and hepatocellular carcinoma. In the trial NCT02466802, we have discovered that regorafenib can be safely combined with the phosphodiesterase 5 inhibitor sildenafil in advanced solid tumor patients. The present studies determined whether the approved ERBB1/2/4 and RAS downregulating drug neratinib, could enhance the lethality of [regorafenib + sildenafil]. Neratinib enhanced [regorafenib + sildenafil] lethality in a greater than additive fashion in colon cancer cells. The drug combination reduced the expression of mutant K-RAS and of multiple histone deacetylase (HDAC) proteins that required autophagosome formation. It caused green fluorescent protein or red fluorescent protein-tagged forms of K-RAS V12 to localize into large intracellular vesicles. Compared with [regorafenib + sildenafil], the three-drug combination caused greater and more prolonged activation of the ATM-AMPK-ULK-1 pathway and caused a greater suppression and prolonged inactivation of mammalian target of rapamycin, AKT, and p70 S6K. Approximately 70% of enhanced lethality caused by neratinib required ataxia-telangiectasia-mutated (ATM)-AMP-dependent protein kinase (AMPK) signaling whereas knockdown of Beclin1, ATG5, FADD, and CD95 completely prevented the elevated killing effect. Exposure of cells to [regorafenib + sildenafil] reduced the expression of the checkpoint immunotherapy biomarkers programmed death-ligand 1, ornithine decarboxylase, and indoleamine 2,3-dioxygenase-1 and increased the expression of major histocompatibility complex A (MHCA), which also required autophagosome formation. Knockdown of specific HDAC proteins recapitulated the effects observed using chemical agents. In vivo, using mouse cancer models, neratinib significantly enhanced the antitumor efficacy of [regorafenib + sildenafil]. Our data support performing a new three drug Phase I trial combining regorafenib, sildenafil, and neratinib.

Keywords: DNA damage; HDAC; autophagy.

Conflict of interest statement

There are no conflicts of interest to report.

© 2018 Wiley Periodicals, Inc.

Figures

Figure 1.. Representative images of proteins knocked down or over-expressed in the present studies.

The data are from PAN02 (mouse) cells and HCT116 (human) cells.

Figure 2.. [Regorafenib + sildenafil] causes concerted signaling to activate toxic PKG signaling.

A. GI tumor cells were treated with vehicle control, regorafenib (0.5 μM), sildenafil (2.0 μM) or the drugs in combination for 2h. Cells were fixed in place and immunostaining performed to determine the staining intensity for VASP-1 S239. At least forty cells per condition were imaged in independent triplicate and plotted as a percentage of sildenafil treatment; no staining for VASP-1 S239 was observed in control treated or regorafenib treated cells (n = 3 +/− SD). # p < 0.05 greater than sildenafil value. B. HEPG2 cells were loaded with DAF-FM DA and were then treated with vehicle control, regorafenib (0.5 μM), sildenafil (2.0 μM) or the drugs in combination for 2h. Data are expressed as a -Fold increase in RNS levels compared to those in vehicle control treated cells (n = 3 +/− SEM) # p < 0.05 greater than vehicle control value; ## p < 0.05 greater than sildenafil value. C. left: HEPG2 cells were treated with vehicle control or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] in combination for 2h. Cells were fixed in place and immunostaining performed to determine the staining intensity for total ATM, P-ATM S1981, total AMPKα, P-AMPKα T172, total γH2AX and P-γH2AX. At least forty cells per condition were imaged in independent triplicate and plotted as a percentage of vehicle treatment (n = 3 +/− SD). # p < 0.05 greater than vehicle control value; Right: HEPG2 cells were treated with vehicle control or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] in combination for 2h. The levels of ATP in treated cells were determined according to manufacturer’s instructions (n = 3 +/− SD). * p < 0.05 less than vehicle control value. D. PANC-1 cells were transfected with a scrambled siRNA (siSCR) or an siRNA to knock down expression of ATM. Twenty-four h later, cells were treated with vehicle control or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] in combination for 2h. Cells were fixed in place and immunostaining performed to determine the staining intensity for total AMPKα and P-AMPKα T172. At least forty cells per condition were imaged in independent triplicate and plotted as a percentage of vehicle treatment (n = 3 +/− SD). * p < 0.05 less than corresponding value in siSCR. E. HCT116 (colorectal) and PAN02 (pancreatic cancer cells were transfected with a scrambled siRNA (siSCR) or an siRNA to knock down expression of AMPKα. Twenty-four h later, cells were treated with vehicle control or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] in combination for 2h. Cells were fixed in place and immunostaining performed to determine the staining intensity for P-eNOS S1177 and P-VASP-1 S239. At least forty cells per condition were imaged in independent triplicate and plotted as a percentage of vehicle treatment (n = 3 +/− SD). # p < 0.05 greater than vehicle control value; ## p < 0.05 greater than corresponding value in siAMPKα cells. F. Cells were transfected with a scrambled siRNA (siSCR) or an siRNA to knock down expression of PKGI and PKGII. Twenty-four h later, cells were treated with vehicle control or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] in combination for 24h. Cells were isolated and viability determined by trypan blue exclusion (n = 3 +/− SD). * p < 0.05 less than corresponding siSCR value.

Figure 3.. Neratinib enhances [regorafenib + sildenafil] lethality which correlates with enhanced K-RAS degradation and inactivation of AKT, mTOR, p70 S6K and ERK1/2.

A. Tumor cells (HCT116, HT29: human colorectal cancer; CT26, MCC38: mouse colorectal cancer) were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 12h. Cells were then isolated, and viability determined by trypan blue exclusion assay (n = 3 +/− SD). B. CT26 cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 4h. Cells were fixed in place at each time point, and immunofluorescent staining performed to determine the total expression of KRAS and total expression of ERK2. At least forty cells per condition were imaged in independent triplicate and the intensity ratio of K-RAS levels to total ERK2 expression plotted as a percentage of control treatment (n = 3 +/− SD). * p < 0.05 less than vehicle control; ** p < 0.05 less than neratinib alone value. Upper images: K -RAS protein become localized in punctate bodies 4h after exposure to neratinib (teal arrows).

Figure 4.. Neratinib and [regorafenib + sildenafil] both cause the rapid intracellular vesicularization of GFP-K-RAS V12 and RFP-K-RAS V12.

A. PANC-1 cells were co-transfected with plasmids to express GFP-K-RAS V12 and RFP-K-RAS V12. Twenty-four h later, cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 30 min, 60 min, 90 min, 120 min and 180 min. Live cells were imaged at each timepoint at 60X magnification. The images presented are representative of three independent experiments. B. In parallel to the studies in Panel A, cells were fixed in paraformaldehyde after 30 min, that preserves the GFP and RFP fluorescence. Cells were immuno-stained to detect the expression of Beclin1 and images taken at 60X magnification. The image presented is representative of three independent experiments.

Figure 5.. Neratinib down-regulates the expression of chaperone proteins.

A. HCT116, SW480 and HT29 cells were treated with vehicle control or with neratinib (50 nM). Cells were fixed in place after 6h and immuno-staining against the chaperones COOH-termini performed to determine the total expression of GRP78 and HSP90. (n = 3, * p < 0.05 less than vehicle control value +/− SEM). B. HCT116 colon cancer cells were treated with vehicle control or with [regorafenib (0.5 μM) + sildenafil (2.0 μM)] for 6h. Cells were fixed in place and immunostaining performed to determine the expression of the indicated growth factor receptors. (n = 3, * p < 0.05 less than vehicle control value +/− SEM).

Figure 6.. Neratinib down-regulates the expression of ERBB1, K-RAS and N-RAS via autophagy.

A. HCT116 colorectal tumor cells were transfected with a scrambled siRNA, with an siRNA to knock down the autophagy regulatory protein Beclin1 (B1), or with an siRNA to knock down the lysosomal protease cathepsin B (CB). After 24h, the expression of Beclin1 or of cathepsin B had been reduced by >75% compared to scrambled control. Twenty-four h after transfection, cells were treated with vehicle control or with neratinib (50 nM) for 0–8h. Vehicle control exposure did not alter receptor basal expression. Cells were fixed in place at the indicated time points and immunofluorescence staining performed to determine the total expression of ERBB1, ERBB2, ERBB3. Cells were visualized in the Hermes WiScan instrument at 10X magnification. The intensity of 40 cells per condition was determined using the machine’s own software combined with data from another two wells of separately treated cells, and with background fluorescence subtracted. Secondary antibody alone staining did not generate any signal (* p < 0.05 less than vehicle control; the data are the mean fluorescence from 120 data points from three separate drug exposures +/− SEM). B. PANC1 (mut. K-RAS), HEPG2 (mut. N-RAS) and HCT116 (mut. K-RAS) cells were transfected with a scrambled siRNA control molecule or siRNA molecules to knock down the expression of Beclin1 or ATG5. Twenty-four h after transfection cells were treated with vehicle control or neratinib (50 nM) for 6h. Cells were fixed in place at the indicated time points and immuno-fluorescence staining performed to determine the total expression of ERBB1, K-RAS & N-RAS. Cells were visualized in the Hermes WiScan instrument at 10X magnification. The staining intensity of 40 cells per condition was determined using the machine’s own software combined with data from another two wells of separately treated cells, and with background fluorescence subtracted. Secondary antibody alone staining did not generate any signal (* p < 0.05 less than vehicle control; the data are the mean fluorescence from 120 data points from three separate drug exposures +/− SEM).

Figure 7.. Neratinib prolongs and enhances the [regorafenib + sildenafil] -induced inactivation of mTOR that is associated with increased expression of ATG5 and Beclin1, and decreased expression of MCL-1 and BCL-XL.

A, B. and C. CT26 (mouse) and HCT116 (human) colon cancer cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 4h and 8h. Cells were fixed in place at each time point, and immunofluorescent staining performed to determine the total expression of ERK2, and the changes in protein phosphorylation and protein expression of the indicated proteins. At least forty cells per condition were imaged in independent triplicate and the intensity ratio of phosphorylation / expression levels to total ERK2 expression determined (n = 3 +/− SD). * p < 0.05 less than values in either individual treatment; # p < 0.05 than values in either individual treatment.

Figure 8.. Death receptor signaling and autophagosome formation are essential for neratinib to enhance [regorafenib + sildenafil] lethality.

A. CT26 cells were transfected with a scrambled control siRNA (siSCR) or were transfected to knock down the expression of the indicated proteins (see Figure 7 for knock down images). Twenty-four h after transfection cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 12h. Cells were then isolated, and viability determined by trypan blue exclusion assay (n = 3 +/− SD). * p < 0.05 greater than reg/sil value in siSCR cells; ** p > 0.05 greater than reg/sil value in siSCR cells. B. CT26 cells were transfected with an empty vector control plasmid (CMV) or were transfected with plasmids to express the indicated proteins: c-FLIP-s; BCL-XL; dominant negative caspase 9 (see Figure 7 for expression images). Twenty-four h after transfection cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 12h. Cells were then isolated, and viability determined by trypan blue exclusion assay (n = 3 +/− SD). * p < 0.05 greater than reg/sil value in siSCR cells; ** p > 0.05 greater than reg/sil value in siSCR cells. C. PANC1, PAN02 and CT26 cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 3h. Cells were fixed in place without membrane permeabilization. Immunofluorescent staining performed to determine the total plasma membrane levels of CD95. At least forty cells per condition were imaged in independent triplicate (n = 3 +/− SD); # p < 0.05 greater than values in [R+S] treatment.

Figure 9.. Exposure of GI tumor cells to [regorafenib + sildenafil + neratinib] reduces the expression of multiple HDAC proteins, that requires autophagosome formation.

A. CT26 cells were treated with vehicle control or with [regorafenib (0.5 μM) + sildenafil (2.0 μM) + neratinib (50 nM)] for 6h. Cells were fixed in place at each time point, and immunofluorescent staining performed to determine the total expression of HDACs1–11. At least forty cells per condition were imaged in independent triplicate (n = 3 +/− SD). * p < 0.05 less than vehicle control. B. and C. PAN02 mouse and PANC-1 human pancreatic cancer cells were transfected with a scrambled control siRNA (siSCR) or with siRNA molecules to knock down the expression of ATG5 or Beclin1. Twenty-four h after transfection cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 6h. Cells were fixed in place and immunofluorescent staining performed to determine the changes in HDAC protein expression of the indicated HDAC proteins. At least forty cells per condition were imaged in independent triplicate (n = 3 +/− SD). * p < 0.05 less than vehicle control.

Figure 10.. Altered expression of HDAC proteins is mechanistically linked to altered expression of immunotherapy biomarker proteins.

A. Colon cancer cells were treated with vehicle control or, as indicated, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or [regorafenib + sildenafil + neratinib] for 6h. Cells were fixed in place and immunofluorescence staining performed to determine the expression of PD-L1, PD-L2, MHCA and IDO-1. At least forty cells per condition were imaged in independent triplicate (n = 3 +/− SD). * p < 0.05 less than vehicle control; # p < 0.05 greater than vehicle control. B. PAN02 cells were transfected with a scrambled control siRNA (siSCR) or with siRNA molecules to knock down the expression of ATG5 or Beclin1. Twenty-four h after transfection cells were treated with vehicle control, [regorafenib (0.5 μM) + sildenafil (2.0 μM)], neratinib (50 nM) or the drugs in combination for 6h. Cells were fixed in place and immunofluorescent staining performed to determine the changes in PD-L1, PD-L2, MHCA, IDO-1 and ODC expression. At least forty cells per condition were imaged in independent triplicate (n = 3 +/− SD). * p < 0.05 less than vehicle control; # p < 0.05 greater than vehicle control. C. PAN02 cells were transfected with a scrambled control siRNA (siSCR) or with siRNA molecules to knock down the expression of HDACs 1, 3, 5, 6, 9, and 11, alone or in combination. Twenty-four h after transfection cells were fixed in place and immunofluorescent staining performed to determine the changes in PD-L1, PD-L2, MHCA, IDO-1 and ODC expression. At least forty cells per condition were imaged in independent triplicate (n = 3 +/− SD). * p < 0.05 less than vehicle control; # p < 0.05 greater than vehicle control.

Figure 11.. Neratinib enhances the ability of [regorafenib + sildenafil] to suppress the growth of colorectal tumors.

A. and B. Female-derived CT26 mouse colorectal / male-derived PAN02 mouse pancreatic carcinoma cells (2 × 104) were implanted into rear flanks of female BALB/c and male C57/BL6 mice, respectively. Tumors were permitted to form until the mean tumor volume was ~40 mm3. Animals were then segregated into groups with near identical mean volumes and the animals then treated for three days with the indicated therapeutic agents: vehicle control (cremophore); regorafenib (20 mg/kg QD Days 1–3); [regorafenib (20 mg/kg QD Days 1–3 and sildenafil (5 mg/kg QD Days 1–3)]; neratinib (15 mg/kg QD Days 1–3); or the three drugs in combination. Tumor volumes were measured prior to drug administration and every three days after the initiation of therapeutic interventions. (n = 10 mice per group +/−SEM). Before, during and after drug treatment tumors are calipered as indicated in the Figure and tumor volume was assessed up to 24 days later. When the volume of the tumor reached >1,500 mm3, animals were humanely sacrificed. * p < 0.05 less growth than tumors treated with [regorafenib + sildenafil]. C. Normal tissues were fixed in formalin on Day 24. Tissues were embedded in paraffin wax and five-micron sections taken. Sections were deparaffinized and H&E stained. Images of the normal tissues were taken at 40X magnification.