Inhibiting the interaction of cMET and IGF-1R with FAK effectively reduces growth of pancreatic cancer cells in vitro and in vivo

Abstract

Pancreatic cancer is one of the most lethal diseases with no effective treatment. Previously, we have shown that FAK is overexpressed in pancreatic cancer and plays a key role in cancer cell survival and proliferation. FAK has been shown to interact with growth factor receptors including cMET and IGF-1R. As a novel therapeutic approach, we targeted the protein interaction of FAK with growth factor receptors to block tumor growth, alter signaling pathways and sensitize cells to chemotherapy. We have selected a small molecule compound (INT2-31) that decreases phosphorylation of AKT via disrupting interaction of FAK with cMET and IGF-1R. Our results demonstrate that interaction of a small molecule compound with FAK decreases phosphorylation of FAK Y397 while increasing FAK Y407 phosphorylation, without inhibiting the kinase activity of FAK and dramatically reduces downstream signaling to AKT. Our lead compound, INT2-31, demonstrates significant inhibition of tumor cell growth in two orthotopic models of pancreatic cancer. In addition, INT2-31 increases sensitivity to gemcitabine chemotherapy in a direct fresh biopsy xenograft model of pancreatic cancer growth.

Figure 1

Structure of INT2-31.

Figure 2. INT2-31 disrupts binding of cMET and IGF-1R with FAK

Panc-1 cells were treated with increasing doses of INT2-31 for one hour duration. An antibody to FAK was utilized to pulldown FAK and coimmunoprecipitation of IGF-1R, cMET and RIP was evaluated (top panel). Western blot analysis following treatment with INT2-31 demonstrates a slight decrease in FAKY397 with an increase in FAK407. This is associated with a decrease in p-Akt and an increase in p-ERK.

Figure 3. INT2-31 increases p-FAK⁴⁰⁷ and decreases p-Akt

Panc-1 and Miapaca-2 cells were treated with INT2-31 for 5, 15, 30 and 60 minutes in full serum. Western blot analysis demonstrates an increase in p-FAK⁴⁰⁷ in Miapaca-2 cells associated with a decrease in p-Akt.

Figure 4. INT2-31 decreases phosphorylation of Y13454 on cMET

Following 24 hours of serum starvation, and 10 min HGF treatment of Panc-1 cells in the presence of increasing doses of INT2-31, phosphorylation of Y1345 on cMETis reduced in a dose dependent manner, whereas Y1230-34-35 remained unchanged. These results indicate that cMET was induced by HGF, yet disruption of its binding to FAK suppressed its full activation.

Figure 5. A peptide corresponding to 16aa of the FAK FERM domain yields similar results to INT2-31

Panc-1 (upper panel) and Miapaca-2 (lower panel) cells were incubated with a 16 aa peptide (SVKAKTLRKLIQQTFR) corresponding to the FAK FERM domain for 12 hours. This resulted in a reduction in p-Y397 FAK and p-S473 AKT, as well as increased p-Y407 FAK, similar to INT2-31 treatment.

Figure 6. Effect of INT2-31 treatment in combination with gemcitabine

Incubation of Panc-1 and Miapaca-2 cells for 48 hours with the combination of gemcitabine (5µM) and INT2-31 (5µM) resulted in a PARP and caspase-9 cleavage.

Figure 7. Effect of in vivo administration of INT2-31 in an orthotopic models of Miapaca-2 cells

Bioluminescent imaging of luciferase expressing Miapaca-2 cells reveals a visual decrease in tumor signal following daily intraperitoneal treatment with 50 mg/kg of INT2-31 as compared to PBS control. Quantification of the photon emission failed to reach statistical significance, possibly due to the large variation in the photon emission from each tumor.

Figure 8. Effect of in vivo administration of INT2-31 in orthotopic models of Panc-1 cells

Bioluminescent imaging of luciferase expressing Panc-1 cells reveals a visual decrease in tumor signal following daily intraperitoneal treatment with 20 mg/kg of INT2-31 as compared to PBS control. Quantification of the photon emission failed to reach statistical significance, possibly due to the large variation in the photon emission from each tumor.

Figure 9. Tumor weights and Ki67 proliferative index in animals implanted with orthotopic Panc-1 cells

Treatment with INT2-31 was associated with a significant decrease in tumor weights (A) and Ki67 proliferative index (B) in those animals treated with INT2-31 compared to PBS control.

Figure 9. Effect of in vivo administration of INT2-31 plus gemcitabine in a direct fresh biopsy xenograft model of pancreatic cancer

Daily IP administration of low dose INT2-31 (20 mg/kg) in combination with gemcitabine (25mg/kg every 3 days) chemotherapy decreased growth of a direct pancreatic cancer xenograft compared to any therapy alone. (* p<0.05 for combination treatment)