Research ArticleExperimental Studies

Murine Double Minute 2 Antagonist Nutlin-3 Enhanced Chemosensitivity in Esophageal Squamous Cell Carcinoma

KEN ITO, HIROTAKA ISHIDA, FUMIYOSHI FUJISHIMA, YASUHIRO NAKAMURA, TAKURO KONNO, KAZUE ISE, SHUKO HATA, YOHEI OZAWA, YUSUKE TANIYAMA, TAKASHI KAMEI and HIRONOBU SASANO
Anticancer Research June 2022, 42 (6) 2875-2882; DOI: https://doi.org/10.21873/anticanres.15769

Abstract

Background/Aim: Murine double minute 2 (MDM2) is well known to inhibit p53 function and its over-expression is associated with poor prognosis in several human malignancies. Nutlin-3, a small-molecule inhibitor of MDM2, exerts antitumor effects on various solid tumors harboring wild-type p53. We aimed to clarify its effects on esophageal cancer. Materials and Methods: We first examined the potential antitumor effects of nutlin-3 according to MDM2 status using esophageal carcinoma cell lines (KYSE 170/180). We then immunolocalized MDM2 immunoreactivity in 62 surgical cases of esophageal squamous cell carcinoma undergoing neoadjuvant chemotherapy followed by esophagectomy and correlated the findings with clinicopathological variables. Results: MDM2 mRNA expression in KYSE 170 was significantly higher than that in KYSE 180 cells. No significant changes were detected in both cell lines when nutlin-3 was added. However, cell proliferation was significantly decreased in KYSE 170 cells treated with nutlin-3 and cisplatin compared to cisplatin alone but not in KYSE 180. MDM2 immunoreactivity was also significantly associated with poor sensitivity to neoadjuvant chemotherapy in the cases examined. Conclusion: The combination of nutlin-3 with chemotherapeutic agents may become a novel therapeutic strategy in esophageal cancer over-expressing MDM2.

Key Words:

Esophageal squamous cell carcinoma is one of the most aggressive gastrointestinal malignancies especially in East Asian countries including Japan (1-4). Results of the Japan Clinical Oncology Group 9907 trial demonstrated neoadjuvant chemotherapy with 5-fluorouracil plus cisplatin followed by esophagectomy with lymph node dissection as the standard treatment approach for patients with localized advanced squamous cell carcinoma (5). However, a poor response to preoperative chemotherapy has been shown to promote tumor development and progression (6). Therefore, the selection of esophageal squamous cell carcinoma patients who could more likely respond to neoadjuvant chemotherapy is clinically required (7, 8).

Murine double minute 2 (MDM2) protein, encoded by the oncogene MDM2, directly binds to p53, resulting in p53 degradation via the addition of ubiquitin (9, 10). The over-expression of MDM2 was reported to be associated with poor prognosis of patients with several human malignancies, including squamous cell carcinoma of the esophagus (11, 12). In addition, clinical trials using MDM2 inhibitors are ongoing in patients with osteosarcoma (13), hepatocellular carcinoma (14), ovarian cancer (15), gastric cancer (16), and leukemia (17).

Nutlin-3 is a novel small-molecule inhibitor of MDM2, considered to exert antitumor effects on solid tumors and lymphoid neoplasms harboring wild-type p53 (16, 17). The antitumor effects of nutlin-3 were also investigated in vitro and in vivo using upper gastrointestinal carcinoma cell lines (16) but its effects on esophageal squamous cell carcinoma cells remain unknown.

Therefore, in this study, we aimed to: 1) clarify the effects of nutlin-3 on the expression of MDM2, and its antitumor effects in combination with or without chemotherapeutic agents, in esophageal carcinoma cell lines; and 2) evaluate the response to preoperative chemotherapy and eventual clinical outcomes of the patients according to MDM2 status using surgically resected specimens of esophageal squamous cell carcinoma.

Materials and Methods

Cell lines and culture. Two human esophageal squamous cell carcinoma cell lines, KYSE 170 and 180, were commercially obtained from the Health Science Research Resources Bank (Tokyo, Japan) and cultured according to the manufacturer’s instructions. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. All cell lines examined were authenticated via short tandem repeat analysis (BEX, Tokyo, Japan).

RNA isolation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions, and complementary DNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). qRT-PCR was performed using a light cycler (Roche, Basel, Switzerland). The primer sequences used in this study were as follows: MDM2 forward: 5’-GGGAAATCTCTGAGAAAGCCAAACTGGAAA-3’ and reverse, 5’-TCCTCAACACATGACTCTCTGGAATCATTC-3’. RPL13A was used as the housekeeping gene for mRNA quantification.

Pharmaceutical reagents. Cisplatin, an inorganic platinum complex, and nutlin-3, a small-molecule inhibitor of MDM2, were both purchased from Wako Pure Chemical Industries (Osaka, Japan). Cisplatin and nutlin-3 were dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions.

Cell proliferation assay. KYSE 170 and 180 cells were seeded in 96-well plates (1×104 cells/well). After 24 h, nutlin-3, cisplatin, or nutlin-3+cisplatin was added individually. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] was added to each well 24 h after the nutlin-3/cisplatin/nutlin-3+cisplatin treatment. The number of cells was counted using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). The absorbance at 450 nm was measured using a microplate reader (Bio-Rad, Tokyo, Japan), and proliferation was evaluated as the average absorbance of each of the six samples. Equivalent volumes of DMSO were used as vehicle controls.

Acquisition of tumor specimens and clinical data. Surgically resected specimens and clinicopathological data were retrieved from 62 patients diagnosed with squamous cell carcinoma of the esophagus at the Department of Pathology, Tohoku University Hospital, Sendai, Japan. All patients underwent neoadjuvant chemotherapy with intravenous cisplatin (80 mg/m2/day on days 1 and 22) and continuous intravenous 5-fluorouracil (800 mg/m2/day from days 1 to 5 and 22-26), followed by radical esophagectomy via thoracoscopy. Histopathological features of all surgical specimens were independently reviewed by three authors (KI, FF, and HS). Tumor, nodes, and metastasis (TNM) staging was performed according to the eighth edition of the American Joint Committee on Cancer/Union for International Cancer Control TNM staging system for esophageal carcinoma (18). The Histopathological tumor regression grade was tentatively classified into five different categories according to the Japanese Classification of Esophageal Cancer, eleventh edition as follows: Grade 3, markedly effective (no viable residual tumor cells); Grade 2, moderately effective (less than one-third residual tumor cells); Grade 1b, slightly effective (one-third to two-thirds residual tumor cells); Grade 1a, slightly effective (more than two-thirds residual tumor cells); and Grade 0, ineffective (no therapeutic effect observed) (19). Grades 0 and 1a were considered as “ineffective” and grades 1b and 2 “effective” (6). Overall survival of patients was tentatively defined as the time from the initial pathological diagnosis to the time of death or the last censor. The exclusion criteria were as follows: 1) the tumor was located at the hypopharynx or the esophagogastric junction and 2) no viable residual carcinoma cells were detected following neoadjuvant chemotherapy (cases of grade 3). The study protocol was approved by the Ethics Committee of Tohoku University (No. 2013–1–474), and informed consent was obtained from all patients examined.

Immunohistochemical analysis. Specimen preparation and immunohistochemistry for MDM2 were performed in all cases as previously described (20-22). The MDM2 antibody used in this study was SMP14 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; diluted 1/1,000) (12). Two of the authors (KI and FF) were blinded to the patients’ clinical information and independently evaluated the results of immunohistochemical analysis. The labeling index of MDM2-positive nuclei was determined for three regions of the deepest area, and 1,000 viable tumor cells were evaluated using microscopy at ×400 magnification. Positive immunoreactivity of MDM2 was defined as a labeling index of ≥20% and negative with an index of <20% (11). We confirmed the accuracy of immunohistochemical staining using external positive controls (breast cancer cells for MDM2) (23).

Statistical analysis. JMP Pro version 16.0.0 software (SAS Institute, Inc., Cary, NC, USA) was used for all statistical analyses. Differences between clinicopathological factors and immunoreactivity were evaluated using Pearson’s χ 2-test or Mann-Whitney U-test. Student’s t-test was used for the analysis of the in vitro study, and statistical significance was set at p<0.05. Overall survival curves were constructed using the Kaplan-Meier method and compared using the log-rank test.

Results

MDM2 mRNA expression levels in KYSE 170/180 cell lines. MDM2 mRNA was detected in KYSE 170/180 cell lines using qRT-PCR assay but the expression of MDM2 mRNA in KYSE 170 was significantly higher than that in KYSE 180 (p<0.001, Figure 1).

Figure 1.
Figure 1.

Quantitative reverse transcription-polymerase chain reaction analysis of MDM2 in esophageal carcinoma cell lines. The expression of MDM2 mRNA in KYSE 170 was significantly higher than that in KYSE 180 (p<0.001).

Effects of cisplatin and nutlin-3 treatment on cell proliferation in esophageal carcinoma cell lines. The cell proliferation rate of the two cell lines (KYSE 170 and 180) treated with 5-20 μM cisplatin was significantly lower than that of the control (p<0.001, Figure 2). KYSE 170 cell lines appeared to have some tolerance to cisplatin compared to KYSE 180 cell lines. No significant changes were detected when 1-10 μM nutlin-3 was added (Figure 3). However, cell proliferation was significantly decreased in KYSE 170 cells treated with cisplatin plus nutlin-3 (cisplatin: 5 or 10 μM, nutlin-3: 1-10 μM) compared to the control (p<0.05, Figure 4A and B). With an exception of the treatment with 5 μM cisplatin and 10 μM nutlin-3, the proliferation ratio in KYSE 180 cells did not decrease compared to the control (Figure 4C and D).

Figure 2.
Figure 2.

The proliferation of KYSE 170 (A) and KYSE 180 (B) after treatment with 5-20 μM cisplatin decreased compared with the control (p<0.001), as shown by the cell proliferation assay.

Figure 3.
Figure 3.

No significant changes in cell proliferation were detected when 1-10 μM nutlin-3 was added to KYSE 170 (A) and KYSE 180 (B) cells.

Figure 4.
Figure 4.

Proliferation was significantly decreased in KYSE 170 cells treated with cisplatin+nutlin-3 (cisplatin: 5 μM, nutlin-3: 1-10 μM) compared with control (p<0.05 for all concentrations) (A). Proliferation was significantly decreased in KYSE 170 cells treated with cisplatin+nutlin-3 (cisplatin: 10 μM, nutlin-3: 1-10 μM) compared with control (p<0.05 for all concentrations) (B). The decreased cell proliferation ratio compared to control was not detected in KYSE 180 cells except after treatment with 5 μM cisplatin+10 μM nutlin-3 (C). No changes in the proliferation ratio compared to control were detected in KYSE 180 cells treated with cisplatin+nutlin-3 (cisplatin: 10 μM, nutlin-3: 1-10 μM) (D).

Clinicopathological features and immunohistochemical characteristics of the cases examined. The correlation between the clinicopathological characteristics of the patients examined in this study and their immunohistochemical profiles are summarized in Table I. According to the histopathological tumor regression grade (19), the patients were tentatively classified as follows: Grade 0, 4 (6%); Grade 1a, 31 (50%); Grade 1b, 21 (34%); and Grade 2, 6 (10%). The mean labeling index of the MDM2 positive cases (33 cases) was 59.6%, ranging from 21.0% to 98.1% (Figure 5). MDM2 immunoreactivity was significantly associated with poor tumor regression grade of the patients (p=0.025). In contrast, no significant differences were detected in the five-year overall survival of the patients harboring high and low MDM2 expression.

View this table:
Table I.

Correlation between MDM2 status and clinicopathological features.

Figure 5.
Figure 5.

Representative images of positive and negative MDM2 immunohistochemical staining. The representative case demonstrated MDM2 immunoreactivity in the nuclei of carcinoma cells (A). The representative case shown here was completely negative for MDM2 immunoreactivity (B) (×400 magnification).

Discussion

In this study, nutlin-3 was shown to enhance the antitumor effects of cisplatin in the esophageal carcinoma cell line KYSE 170. The antitumor effects detected in KYSE 170 were dose-dependent, whereas KYSE 180 cells did not demonstrate these effects. MDM2 mRNA levels in KYSE 170 were significantly higher than those in KYSE 180, indicating that MDM2 expression may be the source of the difference in the effects of nutlin-3 between KYSE 170 and 180. Nutlin-3, could therefore inhibit MDM2 function, leading to increased sensitivity to chemotherapy. Endo et al. reported that the combination of nutlin-3 and 5-fluorouracil significantly inhibited cell proliferation of xenograft tumors compared to each agent alone (16); a cell line over-expressing MDM2 was used in the xenograft experiment. Therefore, the molecular mechanisms mentioned above and the over-expression of MDM2 were considered to play pivotal roles in the nutlin-3-induced synergistic cytotoxicity in combination with cytotoxic agents (16).

Both cell lines used in this study had mutations in TP53. MDM2 inhibited transactivation of tumor suppressor p53, which could explain why nutlin-3 exerted its therapeutic effects on the cell line with mutant p53. Klein et al. recently reported that MDM2 contributed to cell cycle progression in p53-null cancer cells in conjunction with E2F1 and p73 (24). E2F1 is a well-known member of the E2F family of transcription factors that control the cell cycle, apoptosis, DNA damage response, and drug resistance (25-28). E2F1 has been reported to drive chemotherapeutic drug resistance in lung and breast carcinoma cells via the activation of ATP-binding cassette transporter expression (28, 29). p73 is a well-known structural and functional homolog of the tumor-suppressing transcription factor p53 that is rarely mutated in cancer cells, and TAp73, the full-length form, has also been reported to be involved in a p53-like function (30, 31). TAp73 has also been reported to have functions contrasting to p53, such as cell growth, survival, and wound healing (30, 32), and to be required in E2F1 gene transcription as well. In the absence of wild-type p53, MDM2, p73, and E2F1 cooperate in a feedback loop (24). MDM2 has also been reported to promote the activity of E2F1 and p73, resulting in cell cycle progression and drug resistance (24). In addition, the synergistic effects of nutlin-3 via E2F1 and TAp73 on mutant p53 have also been reported in lung cancer, neuroblastoma, malignant peripheral nerve sheath cell tumor and others (33-36). Therefore, nutlin-3 is also considered to be effective on carcinoma cells harboring aberrant p53.

In contrast, nutlin-3 treatment alone did not decrease the proliferation ratio of KYSE 170/180 cells. Arya et al. reported a dose-dependent sensitivity to nutlin-3 in p53 wild-type cell lines of laryngeal squamous cell carcinoma, whereas all the cell lines with mutant p53 exhibited essentially identical growth regardless of the presence or absence of nutlin-3 (37). In addition, Endo et al. revealed that nutlin-3 arrested the cell cycle in the G1 phase and induced apoptosis in gastric carcinoma cell lines expressing wild-type p53 (16). However, these nutlin-3 effects were not detected in the cell lines harboring mutant p53 (16). These results also indicate the disruption of the normal p53–MDM2 interaction (positive feedback of accumulated p53 to MDM2) in carcinoma cells with mutant p53 (16). Therefore, the cytotoxicity induced by nutlin-3+cisplatin detected in this study was considered to be due to the enhanced chemosensitivity caused by nutlin-3, not the cytotoxicity of nutlin-3 itself. In addition, the high percentage of cases with mutant p53 in esophageal squamous cell carcinoma in this study also demonstrated that nutlin-3 alone was not necessarily considered as an effective treatment. However, the combination therapy of nutlin-3 and chemotherapeutic drugs might enhance the antineoplastic effects, which should lead to improved patient prognosis. Further studies are, however, required to clarify the detailed mechanisms of nutlin-3 induced synergistic cytotoxicity in the p53-mutant carcinoma cells. In addition, other antineoplastic agents, such as 5-fluorouracil or docetaxel, are also required to be explored in combination with nutlin-3 for further clarification.

The potential correlation between MDM2 protein expression and patient clinical outcomes has been reported in various neoplasms (32, 38-41) but this is the first study to demonstrate that high MDM2 immunoreactivity in surgically resected specimens is associated with poor tumor regression grade following neoadjuvant chemotherapy. However, no significant difference was detected in the five-year overall survival between the patients with positive and negative MDM2 status. In the patients with esophageal squamous cell carcinoma who underwent definitive chemoradiotherapy, Okamoto et al. reported that the overall survival of patients was improved when the MDM2 levels were lower, and poor chemo-radiosensitivity in the high MDM2 expression group was associated with worse prognosis (11). Furthermore, esophagectomy was performed in all the patients examined in this study. Therefore, high levels of MDM2 expression were considered to be associated with poor chemosensitivity. However, it is also true that the surgery itself could have influenced the clinical outcome of the patients rather than the status of MDM2 expression of carcinoma cells; this issue awaits further investigations for clarification.

This study has the following limitations. Firstly, biopsy specimens of the patients examined in this study could not be obtained for the great majority of the cases. If good chemosensitivity can be predicted using biopsy specimens (lower MDM2 expression), personalized or tailored medication may be possible. In addition, the p53 (wild-type)–MDM2 interaction has been previously well studied; however, the esophageal carcinoma cell lines used in this study had mutated p53. We consider that the results obtained from these cell lines are useful because TP53 somatic mutations have been identified in more than 80% of esophageal squamous cell carcinoma cases (42). As chemical agents may induce immunogenic cell death in esophageal cancer (43), further investigations on local infiltrating immune cells will be also required.

Conclusion

We evaluated the potential antitumor effects of nutlin-3 on esophageal carcinoma cell lines (KYSE170/180). No significant changes were detected when cells were treated with nutlin-3 but cell proliferation was significantly decreased in KYSE 170 cells (high MDM2 expression) treated with nutlin-3 and cisplatin compared to cisplatin alone, indicating that the synergistic cytotoxicity could be attributed to enhanced chemosensitivity caused by nutlin-3, and not the cytotoxicity of nutlin-3 itself. The positive immunoreactivity of MDM2 was significantly associated with poor chemosensitivity of the patients. Therefore, examination of MDM2 immunoreactivity in biopsy specimens prior to treatment could predict chemosensitivity, leading to potential personalized treatment, such as combination chemotherapy with nutlin-3.

Acknowledgements

The Authors are grateful to the staff of the Department of Pathology, Tohoku University Hospital, Sendai, Japan, for their technical assistance. The Authors would like to acknowledge Mr. Katsuhiko Ono and Ms. Yasuko Furukawa (Tohoku University, Sendai Japan) for their excellent technical support. The Authors would also like to thank Editage (www.editage.jp) for English language editing.

Footnotes

  • Authors’ Contributions

    Ken Ito and Fumiyoshi Fujishima conceived the study. Material preparation was performed by Ken Ito, Hirotaka Ishida, Takuro Konno, Yohei Ozawa, and Fumiyoshi Fujishima. Immunohistochemical staining was performed by Ken Ito, Hirotaka Ishida, Takuro Konno, and Kazue Ise. Ken Ito, Fumiyoshi Fujishima, and Hironobu Sasano evaluated morphological features of the neoplasms examined in this study. Ken Ito and Fumiyoshi Fujishima also evaluated the results of immunohistochemistry. Proliferation assay and qRT-PCR were performed by Ken Ito, Kazue Ise, and Shuko Hata. Data collection was performed by Ken Ito, Hirotaka Ishida, Takuro Konno, Yohei Ozawa, Yusuke Taniyama, and Takashi Kamei. The data were analyzed by Ken Ito, Hirotaka Ishida, Fumiyoshi Fujishima, and Yasuhiro Nakamura. Hironobu Sasano and Takashi Kamei supervised the project. The first draft of the manuscript was written by Ken Ito and Hirotaka Ishida, and all Authors commented on the manuscript. Manuscript editing was performed by all Authors. All Authors approved the final version of the manuscript.

  • Conflicts of Interest

    The Authors have no conflicts of interest to disclose in relation to this study.

  • Received March 23, 2022.
  • Revision received April 16, 2022.
  • Accepted April 18, 2022.

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Murine Double Minute 2 Antagonist Nutlin-3 Enhanced Chemosensitivity in Esophageal Squamous Cell Carcinoma
KEN ITO, HIROTAKA ISHIDA, FUMIYOSHI FUJISHIMA, YASUHIRO NAKAMURA, TAKURO KONNO, KAZUE ISE, SHUKO HATA, YOHEI OZAWA, YUSUKE TANIYAMA, TAKASHI KAMEI, HIRONOBU SASANO
Anticancer Research Jun 2022, 42 (6) 2875-2882; DOI: 10.21873/anticanres.15769
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