Targeting altered cancer methionine metabolism with recombinant methioninase (rMETase) overcomes partial gemcitabine-resistance and regresses a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer

ABSTRACT

Pancreatic cancer is a recalcitrant disease. Gemcitabine (GEM) is the most widely-used first-line therapy for pancreatic cancer, but most patients eventually fail. Transformative therapy is necessary to significantly improve the outcome of pancreatic cancer patients. Tumors have an elevated requirement for methionine and are susceptible to methionine restriction. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer to determine the efficacy of recombinant methioninase (rMETase) to effect methionine restriction and thereby overcome GEM-resistance. A pancreatic cancer obtained from a patient was grown orthotopically in the pancreatic tail of nude mice to establish the PDOX model. Five weeks after implantation, 40 pancreatic cancer PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); GEM (100 mg/kg, i.p., once a week for 5 weeks, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); GEM+rMETase (GEM: 100 mg/kg, i.p., once a week for 5 weeks, rMETase: 100 units, i.p., 14 consecutive days, n = 10). Although GEM partially inhibited PDOX tumor growth, combination therapy (GEM+rMETase) was significantly more effective than mono therapy (GEM: p = 0.0025, rMETase: p = 0.0010). The present study is the first demonstrating the efficacy of rMETase combination therapy in a pancreatic cancer PDOX model to overcome first-line therapy resistance in this recalcitrant disease.

Introduction

Pancreatic cancer is a recalcitrant disease. Gemcitabine (GEM) is the most widely-used first-line therapy, but most patients eventually fail. Transformative therapy is necessary to significantly improve the outcome of pancreatic cancer patients.

Altered cancer metabolism is currently being investigated for targets for effective novel therapeutics [1]. A very promising candidate target is the elevated methionine (MET) requirement of cancer cells, termed MET dependence. MET dependence may be the only known general metabolic defect in cancer [2–4]. MET dependence is observed when cancer cells selectively arrest upon MET restriction [2–5]. Tumor MET levels correlate with tumor size [6], further demonstrating the dependence of tumors on MET. MET dependence is due to MET overuse by cancer cells [2,5,7,8]. MET overuse can be observed in the clinic by the efficacy of [¹¹C]MET-PET imaging which gives a very strong signal, since the cancer tissue is taking up much more MET than the surrounding normal tissues [9]. MET restriction selectively arrests cancer cells in late S/G₂ of the cell cycle where the cancer cells become highly-sensitive to cytotoxic chemotherapy [10–15].

MET is sourced mainly from food. However, MET restriction through diets with low protein content does not allow the maintenance of good nutritional status. In addition, reduction of MET levels by dietary intervention is limited since MET is also sourced from the protein breakdown [2]. In order to more effectively target MET-dependence, we previously cloned Psuedomonas putida L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioniniase [rMETase] [EC 4.4.1.11]) in E. coli for large scale industrial production [16–21].

Targeting MET by rMETase arrested growth of cancer cells in vitro and in vivo [21–30]. We previously reported that rMETase, could inhibit tumor growth in patient-derived orthotopic xenograft (PDOX) nude mouse models of melanoma and sarcoma [24–30]. rMETase alone and in combination with a first-line therapy was very effective in the PDOX models [24–30]. For example, rMETase in combination with doxorubicin (DOX) overcame undifferentiated spindle-cell sarcoma (USCS)-resistance to DOX [27,28], which is first line therapy for this disease.

rMETase combined with temozolomide (TEM) [25] was significantly more efficacious than either mono-therapy in a PDOX model of BRAF-V600E mutant melanoma [31–34]. rMETase combined with both tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and cisplatinum (CDDP) eradicated a osteosarcoma PDOX model [29]. Cell-cycle decoy by S. typhimurium A1-R, cell-cycle trap by rMETase and cell kill by cisplatinum CDDP was able to eradicate the metastatic osteosarcoma PDOX [29].

In the present study, we utilized a PDOX nude mouse model of pancreatic cancer to demonstrate that rMETase can overcome GEM-resistance.

Results and discussion

GEM alone could inhibit (P = 0.001), but not arrest the pancreatic cancer PDOX. rMETase alone could also inhibit tumor growth (P = 0.020), but not arrest the pancreatic cancer PDOX. In contrast, the combination of GEM+rMETase could regress the pancreatic cancer PDOX (p < 0.0001) compared to the untreated control. The combination of GEM+rMETase significantly inhibited tumor growth compared to other treatments. On day-14, GEM: p = 0.0006; rMETase: p = 0.0031. On day-21: GEM: p = 0.0215; rMETase: p = 0.0001. On day-28, GEM: p = 0.0004; rMETase: p < 0.0001). The combination of GEM+rMETase was also significantly more effective than other therapies (GEM: p = 0.0025, rMETase: p = 0.0010) on day 35 (Figure 1), demonstrating the durability of the response.

Figure 1.

Drug efficacy on the pancreatic cancer PDOX. Line graphs show tumor volume at each point relative to the initial tumor volume for each condition. **p < 0.01. Error bars: ± SD.

Intra-tumor MET levels decreased after rMETase treatment (p = 0.0060) (Figure 2). This result showed that the pancreatic cancer PDOX is MET dependent and rMETase has potential to deplete tumor MET.

Figure 2.

Intra-tumor MET levels. Bar graphs show intra-tumor MET levels in control (CTR) and rMETase-treated tumors. Error bars: ± SD.

The body weight on each day, compared with day-0, did not significantly differ between any treatment group (Figure 3). There were no animal deaths in any group or untreated control. These results suggest the safety of rMETase and rMETase combination therapy with GEM. Toxicities not indicated by body weight changes may have occurred in the treated groups.

Figure 3.

Effect of treatment on mouse body weight. Bar graphs show mouse body weight in each treatment group at pre- and post-treatment times.

Histologically, the untreated control tumor was mainly comprised of viable cells. In contrast, tumors treated with the combination therapy (GEM+rMETase) showed a great reduction of cancer cells as well as necrosis (Figure 4). It was not possible to determine if all cancer cells were eliminated. GEM-rMETase treatment resulted in the strongest histological effect on the tumors.

Figure 4.

Tumor histology. A. Untreated control. B. Combination treatment with GEM+rMETase. Scale bars: 100 μm.

The present report demonstrates that rMETase combination therapy could inhibit pancreatic cancer PDOX growth at least 35 days without overt toxicity. In the GEM+rMETase group, the tumor volume at day-0 was 181 ± 54 mm³ and at day-35 the tumor volume was 176 ± 80 mm³. In addition, tumor histology indicated the tumor treated with GEM + rMETase was highly necrotic has much fewer, if any, cancer cells compared with the tumor at day-0. These results suggest that GEM + rMETase at day-35 has regressed and potentially cured the tumor. These results indicate that GEM combined with rMETase has future clinical potential. The present study demonstrates the power of the PDOX models to identify highly effective therapy for pancreatic cancer, one of the most recalcitrant cancers. The present study indicates that possibly in the near future more effective therapy will be clinically available for pancreatic cancer, which can be identifiable for individual patients using PDOX models.

Using the technique of surgical orthotopic implantation (SOI), PDOX models have been developed for pancreatic [6,35–38], breast [39], ovarian [40], lung [41], cervical [42], colon [43–45], stomach cancers [46], sarcoma [47–61] and melanoma [25,27,31–33,62], suggesting that improved therapy using rMETase will be developed for all major cancers.

Previously-developed concepts and strategies of highly-selective tumor targeting can take advantage of molecular targeting of tumors, including tissue-selective therapy which focuses on unique differences between normal and tumor tissues [63–68].

Materials and methods

Mice

Athymic nu/nu male nude mice (AntiCancer, Inc., San Diego, CA), 4–6 weeks old, were used in this study. All mice were kept in a barrier facility on a high efficiency particulate arrestance (HEPA)-filtered rack under standard conditions of 12-hour light/dark cycles. The animals were fed an autoclaved laboratory rodent diet [26]. All animal experiments were performed with an AntiCancer Institutional Animal Care and Use Committee (IACUC)-protocol specifically approved for this study and in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under Assurance Number A3873-1. Anesthesia and analgesics were used for all surgical experiments to avoid unnecessary suffering of the mice. Subcutaneous injection of ketamine mixture (a 0.02 ml solution of 20 mg/kg ketamine, 15.2 mg/kg xylazine, and 0.48 mg/kg acepromazine maleate) was used for mice. The response of animals during surgery was monitored carefully to maintain adequate depth of anesthesia. The animals were observed daily and humanely sacrificed by CO₂ inhalation when they met the following criteria: severe tumor burden (more than 20 mm in diameter), prostration, significant body weight loss, difficulty breathing, rotational motion and body temperature drop.

Patient-derived tumor

A patient diagnosed with pancreatic cancer previously had the tumor resected, which was established in nude mice in the MD Anderson Cancer Center. Written informed consent was provided by the patient and the Institutional Review Board (IRB) of MD Anderson Cancer Center approved this experiment [25,26,30–33,69–71].

Surgical orthotopic implantation (SOI)

After nude mice were anesthetized with the ketamine solution described above, a 1–2 cm skin incision was made on the left side abdomen through the skin, fascia and peritoneum and the pancreas was exposed. Surgical sutures (8-0 nylon) were used to implant tumor fragments onto the tail of pancreas to establish the PDOX model. The wound was closed with a 6-0 nylon suture (Ethilon, Ethicon, Inc., NJ, USA) [6,35–38,69–71].

Recombinant methioninase (rMETase) production

Recombinant L-metionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase]) [EC 4.4.1.11] from Pseudomonas putida has been previously cloned and was produced in Escherichia coli (AntiCancer, Inc., San Diego, CA). rMETase is a homotetrameric PLP enzyme of 172-kDa molecular mass [16].

Intra-tumor MET level analysis

At the end of the treatment period, each tumor was sonicated for 30 seconds on ice and centrifuged at 12,000 rpm for 10 minutes. Supernatants were collected and protein levels were measured using the Coomassie Protein Assay Kit (Thermo Scientific, Rockford, IL). Protein levels were calculated from a standard curve obtained with a protein standard, bovine serum albumin (BSA). MET levels were determined with the HPLC procedure described previously [72]. Standardized MET levels were calculated per mg tumor protein [25].

Treatment study design in the PDOX model of pancreatic cancer

PDOX mouse models were randomized into four groups of 10 mice each: untreated control; GEM (100 mg/kg, i.p., once a week for 5 weeks); rMETase (100 units, i.p., 14 consecutive days); GEM+rMETase (GEM: 100 mg/kg, i.p., once a week for 5 weeks, rMETase: 100 units, i.p., 14 consecutive days). Tumor length and width were measured on day 14, 21, 28 and 35. Tumor volume was calculated with the following formula: Tumor volume (mm³) = length (mm) × width (mm) × width (mm) × 1/2. The data are presented as the tumor volume ratio which is defined at the tumor volume at each point relative to the pre-treatment tumor volume.

Histological examination

Fresh tumor samples were fixed in 10% formalin and embedded in paraffin before sectioning and staining. Tissue sections (5 μm) were deparaffinized in xylene and rehydrated in an ethanol series. Hematoxylin and eosin (H&E) staining was performed according to standard protocols. Histological examination was performed with a BHS System Microscope (Olympus Corporation, Tokyo, Japan). Images were acquired with INFINITY ANALYZE software (Lumenera Corporation, Ottawa, Canada) [25,26,30–33].

Statistical analysis

JMP version 11.0 was used for all statistical analyses. Significant differences for continuous variables were determined using the Mann-Whitney U test. Line graphs express average values and error bars show SD. A probability value of P ≤ 0.05 was considered statistically significant.

Dedication

This paper is dedicated to the memory of A. R. Moossa, M.D., Sun Lee, M.D. and Shigeo Yagi, Ph.D.

Disclosure of potential conflicts of interest

K.K., K.M., T.H., T.K. and R.M.H. are unsalaried associates of AntiCancer Inc. There are no other competing financial interests.