The Role of the Carnitine/Organic Cation Transporter Novel 2 in the Clinical Outcome of Patients With Locally Advanced Esophageal Carcinoma Treated With Oxaliplatin

Abstract

Esophageal cancer is the ninth most common malignancy worldwide, ranking sixth in mortality. Platinum-based chemotherapy is commonly used for treating locally advanced esophageal cancer, yet it is ineffective in a large portion of patients. There is a need for reliable molecular markers with direct clinical application for a prospective selection of patients who can benefit from chemotherapy and patients in whom toxicity is likely to outweigh the benefit. The cytotoxic activity of platinum derivatives largely depends on the uptake and accumulation into cells, primarily by organic cation transporters (OCTs). The aim of the study was to investigate the impact of OCT expression on the clinical outcome of patients with esophageal cancer treated with oxaliplatin. Twenty patients with esophageal squamous cell carcinoma (SCC) were prospectively enrolled and surgical specimens used for screening OCT expression level by western blotting and/or immunostaining, and for culture of cancer cells. Sixty-seven patients with SCC who received oxaliplatin and for whom follow-up was available were retrospectively assessed for organic cation/carnitine transporter 2 (OCTN2) expression by real time RT-PCR and immunostaining. OCTN2 staining was also performed in 22 esophageal adenocarcinomas. OCTN2 function in patient-derived cancer cells was evaluated by assessing L-carnitine uptake and sensitivity to oxaliplatin. The impact of OCTN2 on oxaliplatin activity was also assessed in HEK293 cells overexpressing OCTN2. OCTN2 expression was higher in tumor than in normal tissues. In patient-derived cancer cells and HEK293 cells, the expression of OCTN2 sensitized to oxaliplatin. Patients treated with oxaliplatin who had high OCTN2 level in the tumor tissue had a reduced risk of recurrence and a longer survival time than those with low expression of OCTN2 in tumor tissue. In conclusion, OCTN2 is expressed in esophageal cancer and it is likely to contribute to the accumulation and cytotoxic activity of oxaliplatin in patients with esophageal carcinoma treated with oxaliplatin.

Keywords: OCTN2; biomarker; carnitine transporter; esophageal cancer; oxaliplatin.

FIGURE 1

Expression and function of OCTN2 in tumor tissues and in the respective primary cancer cells. Representative immunostaining of tumor tissue and paired normal mucosa. Scale bar = 100 µm (A). Western blot image of total lysates (20 µg) from 15 tumor samples and the respective surrounding normal tissues probed with anti-OCTN2 and anti-β-actin antibodies (B). Representative immunostaining of OCTN2 in primary patient-derived cancer cells and paired normal mucosa. OCTN2 staining is shown in green; nuclei are stained with 4,6-diamidino2-phenylindole (DAPI) in blue. Original magnification, 200 × (C). Five-minute uptake of L-carnitine in primary patient-derived cancer cells in the presence or absence of Na⁺ (D). Na⁺-dependent uptake of L-carnitine in OCTN2 high-expressing and low-expressing patient-derived cancer cells, according to the OCTN2 staining score western blot quantification (E). Each data point represents the uptake values in cells isolated from one tumor tissue and shown as the mean ± S.D. from three independent experiments. Statistical comparison was performed by unpaired Student’s t-test.

FIGURE 2

Cell growth and oxaliplatin sensitivity in OCTN2 expressing cells. Cells were grouped according to the tissue staining score and western blot quantification. BrdU incorporation in the DNA of OCTN2 high- and low-expressing cancer cells (A). Representative image of TUNEL staining ((B), top panel) of high- and low-expressing cancer cells after 48-h exposure to oxaliplatin at the extracellular concentration of 100 µM. Representative image electron microscopy ((B), bottom panel) of patient-derived cancer cells after 48-h exposure to oxaliplatin at the extracellular concentration of 100 μM at 10,000x. Quantification of the number of apoptotic cells stained in purple normalized for the number of nuclei, stained with 4,6-diamidino2-phenylindole (DAPI) in blue (C). ATP content in OCTN2 high- and low-expressing patient-derived cancer cells after 48-h exposure to oxaliplatin at the extracellular concentration of 100 µM (D). ATP content in HEK-WT and HEK-OCTN2 exposed for 48 h to increasing extracellular concentrations of oxaliplatin (E). All data are presented as mean ± S.D. from three independent experiments and compared by unpaired Student’s t-test. Curves were modelled using the (log (Inhibitor) vs normalized response-variable slope) equation.

FIGURE 3

OCTN2 expression level in squamous cell carcinoma (SCC) and adenocarcinoma (AC) samples. OCTN2 mRNA values normalized by the expression of the housekeeping gene b-actin (A). Representative immunostaining of SCC and AC and the respective paired normal tissues. Scale bar = 100 µm (B). Relative quantification of the OCTN2 staining per high-power field. All comparisons were performed by Mann-Whitney test (C).

FIGURE 4

Clinical outcome of patients with high and low expression of OCTN2 treated with oxaliplatin. Kaplan -Meier progression-free survival and overall survival curves in OCTN2 low- and high-expressing groups defined according to the relative mRNA level (A and B) or the staining score (C and D). Samples with relative mRNA value or staining score ≥ of the median value were classified as OCTN2 high-expressing tumors.

FIGURE 5

PFS of patients with high and low expression of OCTN2 with negative lymph nodes treated with oxaliplatin. Kaplan -Meier progression-free survival curves in lymph node negative patients with low or high mRNA (A) or protein (B) expression of OCTN2. Samples with relative mRNA value or staining score ≥ of the median value were classified as OCTN2 high-expressing tumors.