Targeting methionine with oral recombinant methioninase (o-rMETase) arrests a patient-derived orthotopic xenograft (PDOX) model of BRAF-V600E mutant melanoma: implications for chronic clinical cancer therapy and prevention

ABSTRACT

The elevated methionine (MET) use by cancer cells is termed MET dependence and may be the only known general metabolic defect in cancer. Targeting MET by recombinant methioninase (rMETase) can arrest the growth of cancer cells in vitro and in vivo. We previously reported that rMETase, administrated by intra-peritoneal injection (ip-rMETase), could inhibit tumor growth in a patient-derived orthotopic xenograft (PDOX) model of a BRAF-V600E mutant melanoma. In the present study, we compared ip-rMETase and oral rMETase (o-rMETase) for efficacy on the melanoma PDOX. Melanoma PDOX nude mice were randomized into four groups of 5 mice each: untreated control; ip-rMETase (100 units, i.p., 14 consecutive days); o-rMETase (100 units, p.o., 14 consecutive days); o-rMETase+ip-rMETase (100 units, p.o.+100 units, i.p., 14 consecutive days). All treatments inhibited tumor growth on day 14 after treatment initiation, compared to untreated control (ip-rMETase, p<0.0001; o-rMETase, p<0.0001; o-rMETase+ip-rMETase, p<0.0001). o-rMETase was significantly more effective than ip-rMETase (p = 0.0086). o-rMETase+ip-rMETase was significantly more effective than either mono-therapy: ip-rMETase, p = 0.0005; or o-rMETase, p = 0.0367. The present study is the first demonstrating that o-rMETase is effective as an anticancer agent. The results of the present study indicate the potential of clinical development of o-rMETase as an agent for chronic cancer therapy and for cancer prevention and possibly for life extension since dietary MET reduction extends life span in many animal models.

KEYWORDS:

• Recombinant methioninase
• methionine dependence
• oral administration
• pyridoxal-L-phosphate
• melanoma
• PDOX
• nude mice
• orthotopic

Introduction

Metastatic melanoma is a recalcitrant cancer, with a 5-y survival rate of 7–30% with no cure for stage III and IV melanoma [1–5]. Vitamin D and its receptors play a critical role in signaling in melanoma progression [6].

An excessive requirement for methionine, termed methionine dependence, appears to be a general metabolic defect in cancer [7]. We have previously shown that cancer-cell growth can be selectively arrested by methionine deprivation, such as with recombinant methioninase (rMETase) [7].

In previous studies, we established patient-derived orthotopic xenograft (PDOX) nude mouse model of BRAF V600E-mutant melanoma [8–12]. In this model, we determine the efficacy of rMETase in combination with a first-line melanoma drug, temozolomide (TEM) [11]. The combination therapy of TEM, first-line therapy, and rMETase was significantly more efficacious than either mono-therapy [11].

The present study demonstrates that oral rMETase (o-rMETase) is highly effective in the BRAF-V600E mutant melanoma PDOX.

Results and discussion

Melanoma-PDOX nude mice were randomized into four groups of 5 mice each: untreated control; ip-rMETase (100 units, i.p., 14 consecutive days); o-rMETase (100 units, p.o., 14 consecutive days); o-rMETase+ip-rMETase (100 units, p.o.+100 units, i.p., 14 consecutive days). All treatments inhibited tumor growth on d 14 after treatment initiation, compared to untreated control (ip-rMETase, p<0.0001; o-rMETase, p<0.0001; o-rMETase+ip-rMETase, p<0.0001). o-rMETase was significantly more effective than ip-rMETase (p = 0.0086). The combination of o-rMETase+ip-rMETase was significantly more effective than either monotherapy therapies: ip-rMETase, p = 0.0005; or o-rMETase, p = 0.0367 (Figures 1 and 2).

Figure 1. Photographs of representative tumors from the untreated control and treatment groups on the BRAF V600E mutant-melanoma PDOX. Tumors were resected on d 15 of treatment. Scale bar: 5 mm

Figure 2. Quantitative efficacy of ip-rMETase, o-rMETase and the combination of ip-rMETase and o-rMETase on the BRAF V600E mutant melanoma PDOX. Line graphs show relative tumor volume at each point relative to the initial tumor volume. ##p < 0.01, #p < 0.05. Error bars: ± SD.

Post-treatment plasma MET levels significantly decreased compared to untreated control: ip-rMETase, p = 0.0122; o-rMETase, p = 0.003; o-rMETase+ip-rMETase, p<0.0001 (Figure 3).

Figure 3. Effect of ip-rMETase and o-rMETase on plasma rMETase levels. Bar graphs show plasma MET levels in each group at post-treatment. ##p < 0.01, #p < 0.05. Error bars: ± SD.

Body weight loss was not observed in any treatment group (Figure 4). There were no animal deaths in any group.

These results showed the safety of o-rMETase and its potential for chronic cancer treatment in the clinic.

Figure 4. Effect of ip-rMETase, o-rMETase and the combination of ip-rMETase and o-rMETase on the BRAF V600E mutant-melanoma PDOX mouse body weight. Bar graphs show mouse body weight in each treatment group at pre- and post-treatment. There were no significantly differences between any treatment group and untreated control.

MET dependence is a general metabolic defect in cancer. Methionine dependence is due to excess use of MET for aberrant transmethylation reactions, termed the “Hoffman effect”, analogous to the Warburg effect for elevated glucose use in cancer [7,13–18]. The excessive and aberrant use of MET in cancer is readily observed in [¹¹C]MET PET imaging, where high uptake of [¹¹C]MET results in a very strong and selective tumor signal compared with normal tissue background. [¹¹C]MET is superior to [¹⁸C] fluorodeoxyglucose (FDG) for PET imaging, suggesting MET dependence is more tumor-specific than glucose dependence [19,20].

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 sourced from protein breakdown [7]. Further reduction of plasma MET, and thereby tumor MET, has been achieved with the use of rMETase [11,21–25].

Our laboratory cloned Pseudomonas putida rMeTase into E. coli for large scale production [26]. It has been demonstrated that MET deprivation arrests growth and induces a tumor-selective G₂-phase cell-cycle arrest of cancer cells in vitro and in vivo [27–30].

We reported recently on the efficacy of rMETase against Ewing's sarcoma in a PDOX model. rMETase effectively reduced tumor growth compared to untreated control. Serum and tumor met levels were lower in the rMETase group [25].

In a previous study, the BRAF V600E-mutant melanoma PDOX was sensitive to rMETase and increased TEM efficacy in combination [11].

The present study reports the surprising result that o-rMETase is effective against the BRAF-V600E mutant melanoma PDOX and is more effective than ip-rMETase. The use of o-rMETase opens many possibilities that appear non-toxic for chronic cancer treatment, for cancer prevention and for general life span extension since MET is also a target of aging [31,32]. A future new study will focus on effects of oral methioninase on tumor histology.

The present study demonstrates the power of the PDOX model to identify effective therapy for recalcitrant cancer. Toward this goal of precision personalized oncology, our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI), including pancreatic [33–36], breast [37], ovarian [38], lung [39], cervical [40], colon [41–43] and stomach cancer [44], sarcoma [45–49] and melanoma [8–11,50].

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 [51–56].

Materials and methods

Mice

Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA), 4–6 wk old, were used in this study. Mice were housed in a barrier facility in a high efficacy particulate arrestance (HEPA)-filtered rack under standard conditions of 12-hour light/dark cycles. The animals were fed an autoclaved laboratory rodent diet. All animal studies were conducted 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. All mouse surgical procedures and imaging were performed with the animals anesthetized by subcutaneous injection of a ketamine mixture (0.02 ml solution of 20 mg/kg ketamine, 15.2 mg/kg xylazine, and 0.48 mg/kg acepromazine maleate). The response of animals during surgery was monitored to ensure adequate depth of anesthesia. The animals were observed on a daily basis and humanely sacrificed by CO₂ inhalation if they met the following endpoint criteria: severe tumor burden (more than 20 mm in diameter), prostration, significant body-weight loss, difficulty breathing, rotational motion or body temperature drop [11].

Patient-derived tumor

A 75-y old female patient was previously diagnosed with a BRAF-V600E melanoma of the right chest wall. The tumor was previously resected in the Department of Surgery, University of California, Los Angeles (UCLA). Written informed consent was provided by the patient, and the Institutional Review Board (IRB) of UCLA approved this experiment [8–11].

Establishment of PDOX models of melanoma by surgical orthotopic implantation (SOI)

Subcutaneously-grown BRAF V600E mutant melanoma was harvested and cut into small fragments (3 mm³). After nude mice were anesthetized with the ketamine solution described above, a 5-mm skin incision was made on the right chest into the chest wall, which was split to make space for the melanoma tissue fragment. A single tumor fragment was implanted orthotopically into the space to establish the PDOX model. The wound was closed with a 6-0 nylon suture (Ethilon, Ethicon, Inc., NJ, USA) [8–11].

Recombinant methionase (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 [26].

Formulation of o-rMETase and pyridoxal-L-phosphate (PLP) supplement

Mouse drinking water contained 100 µmol/l PLP. PLP (1.0 ml of 20 mmol/l) was added to 200 ml drinking water and made fresh daily. rMETase was administered daily by gavage using a stainless feeding needle (100 units, 2.0 mg) in phosphate-buffered saline (PBS).

Treatment study design in the PDOX model of melanoma

BRAF V600E mutant melanoma PDOX nude mice were randomized into four groups of 5 mice each: untreated control; ip-rMETase (100 units, i.p., 14 consecutive days); o-rMETase (100 units, p.o., 14 consecutive days); o-rMETase+ip-rMETase (100 units, p.o.+100 units, i.p., 14 consecutive days).

Determination of Plasma methionine

The plasma methionine concentration was measured using a precolumn derivatization, followed by high-performance liquid chromatography separation based on a previously described method with modification [57]. A 10-μl plasma sample or methionine standard was used. The plasma methionine was identified by the retention time of a methionine standard curve. The limit of detection was 0.5 μM methionine. The upper limit of detection for methionine for methionine assay is 100μM.

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 bar show SD. A probability value of P ≤ 0.05 was considered statistically significant.

 Disclosure of potential conflicts of interest

 No potential conflicts of interest were disclosed.