A novel RET inhibitor with potent efficacy against medullary thyroid cancer in vivo

Abstract

Background: Most medullary thyroid carcinomas (MTC) recur or progress despite curative resection. Current targeted therapies show promise but lack durable efficacy and tolerability. The purpose of this study was to build on previous in vitro work and evaluate withaferin A (WA), a novel RET inhibitor, in a metastatic murine model of MTC.

Methods: A total of 5 million DRO-81-1 human MTC cells injected in the left posterior neck of nu/nu mice generated metastases uniformly to the liver, spleen, and/or lungs. Treatment with WA (8 mg/kg/day, intraperitoneally, for 21 days) was started for neoplasms > 100 mm(3). Endpoints were survival, neoplasm > 15,00 mm(3), decreased body weight, or body score (all measured three times/wk).

Results: All controls (saline; n = 5) died or deteriorated from metastatic disease by 7 weeks postinjection. All treated animals were alive (WA; n = 5), having tumor regression and growth delay without toxicity or weight loss at 6 weeks posttreatment (P < .01). Tumor cells treated with WA demonstrated inhibition of total and phospho-RET levels by Western blot analysis in a dose-dependent manner (almost complete inhibition with treatment of 5 μM WA) as well as potent inhibition of phospho-ERK and phospho-Akt levels.

Conclusion: WA is a novel natural-product RET-inhibitor with efficacy in a metastatic murine model of MTC. Further long-term efficacy/toxicity studies are warranted to evaluate this compound for clinical translation.

Figure 1. Neck Xenografts of DRO81-1 MTC cells in Nu/Nu mice develop metastatic disease similar to human MTCs

MTC xenografts in NU/NU mice were created by injection in the left posterior neck with 5 million DRO81-1 cells. All of the control animals (n=5) developed progressively enlarging tumor masses with increased disease burden resulting in deterioration of bodyweight (>10% loss from baseline) as well as decline in body score within 6–7 weeks post-injection leading to natural death or euthanasia as per standard animal protocol monitoring. A representative control animal at autopsy is demonstrated in Figure 1A with a view of both the primary tumor implant as well as intra-abdominal metastases. This xenograft model reproducibly metastasizes to the liver, lungs and spleen in a similar pattern to human MTC. A gross comparison of three of these 3-week post-treatment (5-weeks post-injection of tumor cells) animals is shown in Figure 1B. The control animal on the left has a large primary tumor with evidence of weight-loss (spine more prominent) while the treated animals (2 on the right) have almost completely regressed tumors with no clinical toxicity as evidenced by normal weight, body score and activity levels.

Figure 1. Neck Xenografts of DRO81-1 MTC cells in Nu/Nu mice develop metastatic disease similar to human MTCs

MTC xenografts in NU/NU mice were created by injection in the left posterior neck with 5 million DRO81-1 cells. All of the control animals (n=5) developed progressively enlarging tumor masses with increased disease burden resulting in deterioration of bodyweight (>10% loss from baseline) as well as decline in body score within 6–7 weeks post-injection leading to natural death or euthanasia as per standard animal protocol monitoring. A representative control animal at autopsy is demonstrated in Figure 1A with a view of both the primary tumor implant as well as intra-abdominal metastases. This xenograft model reproducibly metastasizes to the liver, lungs and spleen in a similar pattern to human MTC. A gross comparison of three of these 3-week post-treatment (5-weeks post-injection of tumor cells) animals is shown in Figure 1B. The control animal on the left has a large primary tumor with evidence of weight-loss (spine more prominent) while the treated animals (2 on the right) have almost completely regressed tumors with no clinical toxicity as evidenced by normal weight, body score and activity levels.

Figure 2. Withaferin A has in vivo efficacy against DRO 81-1 xenografts without toxicity

Drug efficacy analysis is plotted out for 8 weeks post-initial tumor injection (since all of the control animals (n=5) have died by 7 weeks post-injection and all of the treated animals (n=5) are alive with tumor). Comparatively even in this small group, treated animals demonstrated a significant survival advantage at this time-point over controls (p<0.01). Tumor growth progression is demonstrated in Figure 2A depicted as an average tumor volume for each group by week (average of all 5 animals per group) with standard error bars. Survival times are depicted in Figure 2B using a Kaplan-Meier format. As a group the treated animals demonstrated a uniform delay-in growth of tumors with 80% of animals demonstrating an initial significant regression in tumor volume at 2 weeks into therapy (week 4 post-tumor implantation) which was followed by eventual disease progression, albeit at a 3 to 4-week growth-delay compared to controls and at a lower growth rate trajectory.

Figure 2. Withaferin A has in vivo efficacy against DRO 81-1 xenografts without toxicity

Drug efficacy analysis is plotted out for 8 weeks post-initial tumor injection (since all of the control animals (n=5) have died by 7 weeks post-injection and all of the treated animals (n=5) are alive with tumor). Comparatively even in this small group, treated animals demonstrated a significant survival advantage at this time-point over controls (p<0.01). Tumor growth progression is demonstrated in Figure 2A depicted as an average tumor volume for each group by week (average of all 5 animals per group) with standard error bars. Survival times are depicted in Figure 2B using a Kaplan-Meier format. As a group the treated animals demonstrated a uniform delay-in growth of tumors with 80% of animals demonstrating an initial significant regression in tumor volume at 2 weeks into therapy (week 4 post-tumor implantation) which was followed by eventual disease progression, albeit at a 3 to 4-week growth-delay compared to controls and at a lower growth rate trajectory.

Figure 3. Withaferin A inhibits RET protooncogene in human medullary thyroid cancer cells in vitro

Human medullary thyroid cancer TT cells and DRO 81-1 cells were treated with increasing concentration of withaferin A for 24 hr and activity (measured as RET-phosphorylation) and total RET proto-oncogenes levels were analyzed using specific antibodies. Membranes were stripped and reprobed with anti-beta actin antibody to ensure equal protein loading and quantitative densiometry is shown below to compare protein expression as a normalized ratio to beta-actin levels. Withaferin A strongly inhibited Tyr905 phosphorylation of Ret at 3.0 uM concentration in TT cells and it reduced expression of total RET protein expression. In DRO 81-1 cells, both phospho-RET and total RET expression are inhibited at 5μM WA concentrations. Both cell lines demonstrate that Withaferin A is functional RET inhibitor in MTC cells.

Figure 4. Inhibition of RET phosphorylation and total RET protein expression by WA in vivo

In ex-vivo tumors removed from Nu/Nu mice with DRO81-1 xenograft injections of 5 million cells to the left posterior neck, prosurvival proteins were analyzed by Western Blot analysis and demonstrate in the WA-treated animals both inhibition of phospho-RET and total RET with beta-actin levels demonstrating equal protein loading in lanes. The dose of WA used in the in vivo model is equivalent to 3 μM in vitro dosing. Quantitative densiometry is shown below to compare protein expression as a normalized ratio to beta-actin levels.

Figure 5. Withaferin A modulates proliferative pathways in medullary thyroid cancer cells in vitro and in vivo

DRO 81-1 cells were treated in vitro with increasing concentration of withaferin A for 24 hr (left western blot) and levels of phosphorylated ERK1/2, total ERK1/2, phosphorylated Akt and total Akt were examined using specific antibodies. Membranes were stripped and reprobed with anti-beta actin antibody to ensure equal protein loading. Quantitative densiometry is shown below to compare protein expression as a normalized ratio to beta-actin levels. These data demonstrate that withaferin A in a concentration-dependent fashion inhibited ERK1/2 phosphorylation and activity as well as total ERK2 levels. In addition withaferin A inhibited Akt activity and reduced total Akt levels. In ex-vivo tumors removed from Nu/Nu mice with DRO81-1 xenograft injections of 5 million cells to the left posterior neck, prosurvival proteins were analyzed by Western Blot analysis and demonstrate in the WA-treated animals reduction of uncleaved caspase 3 (right blot) indicating its cleavage as a marker of apoptotic cell-death. Mechanistically WA has been shown previously in vitro to induce apoptosis in DRO-81-1 cells. These results confirm that the drug is acting through similar pathway mechanisms in vivo as it is in vitro.

Figure 6. Withaferin A treatment significantly reduces xenograft MTC tumor calcitonin production in vivo

DRO 81-1 xenograft nu/nu mice were treated with withaferin A (5.0 mg/Kg/day) or with vehicle. Twenty-one days after treatment, animals were sacrificed to collect tissue samples and blood. Mouse serum calcitonin levels were measured using The Calcitonin Immunoassay from ALPCO Diagnostics (Salem, NH) using manufacturer controls. Graph is average of 3 animal samples with standard deviation error bars. WA treated animals had a significantly lower calcitonin (79 pg/mL) compared to controls (149 pg/mL; p<0.05) indicating the drug’s effect on tumor biochemical functionality.