MCT4 as a potential therapeutic target for metastatic gastric cancer with peritoneal carcinomatosis

Abstract

Monocarboxylate transporters (MCTs) play a major role in up-regulation of glycolysis and adaptation to acidosis. However, the role of MCTs in gastric cancer (GC) is not fully understood. We investigated the potential utilization of a new cancer therapy for GC. We characterized the expression patterns of the MCT isoforms 1, 2, and 4 and investigated the role of MCT in GC through in vitro and in vivo tests using siRNA targeting MCTs. In GC cell lines, MCT1, 2, and 4 were up-regulated with different expression levels; MCT1 and MCT4 were more widely expressed in GC cell lines compared with MCT2. Inhibition of MCTs by siRNA or AR-C155858 reduced cell viability and lactate uptake in GC cell lines. The effect of inhibition of MCTs on tumor growth was also confirmed in xenograft models. Furthermore, MCT inhibition in GC cells increased the sensitivity of cells to radiotherapy or chemotherapy. Compared with normal gastric tissue, no significant alterations of expression levels in tumors were identified for MCT1 and MCT2, whereas a significant increase in MCT4 expression was observed. Most importantly, MCT4 was highly overexpressed in malignant cells of acsites and its silencing resulted in reduced tumor cell proliferation and lactate uptake in malignant ascites. Our study suggests that MCT4 is a clinically relevant target in GC with peritoneal carcinomatosis.

Keywords: gastric cancer; glycolysis; monocarboxylate transporter; prognosis.

Figure 1. Expression levels of MCTs (MCT1, MCT2, and MCT4) in gastric cancer cell lines

RT-PCR and western blot analysis were used to assess MCT expression in 16 gastric cell lines (primary, N = 6; metastasis, N = 4; ascites, N = 6). mRNA levels were normalized to GAPDH and protein levels to β-actin.

Figure 2. Effect of MCT inhibition on cell proliferation and lactate uptake

A. Expression of MCTs in GC cell lines after transfection with siRNAs was analyzed by western blot analysis using antibodies to the proteins indicated. B. Cell proliferation was measured 72 hours after transfection with MCT siRNA (siMCT1, siMCT2, or siMCT4) or a negative control sequence (siC). The percentage of viable cells is shown relative to that of the untreated control. C. [¹⁴C]-L-lactate uptake was measured after knockdown of MCTs in GC cell lines. D. The effect of MCT inhibition on lactate uptake in the SNU668 cell line was evaluated over time and siC was used as a reference. Values represent the mean of 3 independent experiments. Data represent mean and standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. In vivo efficacy of MCT inhibition

BALB/c nude mice were injected subcutaneously with GC cell lines. One week after injection, mice were treated 3 times per week with an intraperitoneal injection of 1 mg/kg AR-C155858. Upper panels show the time course of the growth and lower panels represent the mean tumor volume and standard deviation of SNU668 A. MKN1 B. and SNU620 C. tumors following administration of AR-C155858 (vs. PBS as a control). D. Proliferation in each type of xenograft (SNU668, MKN1, and SNU620) was analyzed by IHC with anti-PCNA antibody (Santa Cruz Biotechnology, PC-10, 1:2000). The brown staining in the nucleus is the PCNA signal. E. For the peritoneal dissemination model, mice were inoculated intraperitoneally with SNU668 cells. The upper panel shows the representative xenograft tumors resected on day 21, showing the difference in tumor volumes. Lower panels represent mean tumor volume and standard deviation. This experiment was repeated three times with similar results. *P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 4. Synergism effect of MCT inhibition with anti-cancer therapy

SNU668 and SNU216 cells were transfected with the indicated siRNAs or treated with AR-155858. Twenty-four hours later, the cells were irradiated with a ¹³⁷Cs source A. and B. or 5-fluorouracil C. Surviving fractions following the given treatments were calculated based on the survival of nonirradiated cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 5. MCTs expression in GC tissues

A. MCT1, MCT2, and MCT4 mRNA and 18S rRNA were detected using real-time PCR according to tissue (normal tissue, tumor tissue, and PDCs collected from malignant ascites). Data were normalized using 18S rRNA as an endogenous control. B. MCT4 immunostaining of gastric cancer tissues and PDCs. Intensity MCT4 immunostatining was measured as score 0 (negative), score 1 (weak), score 2 (moderate), and score 3 (strong) for positive cases of gastric cancer tissue (left panel). Right panel represents two cases of PDCs with highly strong MCT4 expression. Original magnification, x 400. C. Three sets of MCT expression in patient-matched tissue. D. MCT4 expression in PDCs collected from malignant ascites and the effects of its silencing on cell proliferation and lactate uptake. * P < 0.05, ** P < 0.01, *** P < 0.001.