Notch1 is a 5-fluorouracil resistant and poor survival marker in human esophagus squamous cell carcinomas

Abstract

Notch signaling involves the processes that govern cell proliferation, cell fate decision, cell differentiation and stem cell maintenance. Due to its fundamental role in stem cells, it has been speculated during the recent years that Notch family may have critical functions in cancer stem cells or cancer cells with a stem cell phenotype, therefore playing an important role in the process of oncogenesis. In this study, expression of Notch family in KYSE70, KYSE140 and KYSE450 squamous esophageal cancer cell lines and virus transformed squamous esophageal epithelial cell line Het-1A was examined by quantitative RT-PCR. Compared to the Het-1A cells, higher levels of Nocth1 and Notch3 expression in the cancer cell lines were identified. Due to the finding that NOTCH3 mainly mediates squamous cell differentiation, NOTCH1 expression was further studied in these cell lines. By Western blot analyses, the KYSE70 cell line which derived from a poorly differentiated tumor highly expressed Notch1, and the Notch1 expression in this cell line was hypoxia inducible, while the KYSE450 cell line which derived from a well differentiated tumor was always negative for Notch1, even in hypoxia. Additional studies demonstrated that the KYSE70 cell line was more 5-FU resistant than the KYSE450 cell line and such 5-FU resistance is correlated to Notch1 expression verified by Notch1 knockdown experiments. In clinical samples, Notch1 protein expression was detected in the basal cells of human esophagus epithelia, and its expression in squamous cell carcinomas was significantly associated with higher pathological grade and shorter overall survival. We conclude that Notch1 expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro and is significantly associated with a poor survival in human esophageal squamous cell carcinomas.

Figure 1. Quantitative RT-PCR of Notch family in the squamous esophageal cancer cell lines KYSE70, KYSE140 and KYSE450 and the virus transformed normal squamous esophageal epithelial cell line Het-1A (A).

Each PCR was performed twice with almost identical values. Notch1 protein expression was examined by Western blotting (B), showing strong Notch1 in KYSE70 cells, negative in KYSE450 cells and weak positive in both KYSE140 and Het-1A cells. Conventional RT-PCR shows rather the same intensity PCR bands for both KYSE70 and KYSE450 cells with the two primer pairs (C).

Figure 2. Cell growth curves show that both cell lines are growth-inhibited under 1% O² than under 20% O².

KYSE70 cells tolerate hypoxia better than KYSE450 cells.

Figure 3. Hypoxia induction of Notch1 and other stemness factors in vitro.

Notch1 is absent in the KYSE450 cells, but positive in KYSE70 cells under normoxia and even high level of Notch1 expression in these KYSE70 cells under hypoxia is shown. There are weak Hif-1α expression under normoxia and weak Hif-1α induction under hypoxia in KYSE450 cells. Comparably higher levels of expression and Hif-1α induction in KYSE70 cells are shown. Positivity and hypoxia induction of Hif-2α, Sox2, Oct3/4 and Hes-1 in KYSE450 cells and relatively stronger positivity and higher levels of hypoxia induction of these factors in KYSE70 cells are displayed.

Figure 4. KYSE70 cells tolerate higher concentrations of 5-FU than KYSE450 cells under normoxia, and such tolerance difference is even significantly apparent under hypoxia.

Figure 5. Notch1 siRNA knockdown validation in KYSE70 cells (5A).

KYSE70 control means blank KYSE70 cells without any treatment. KYSE70 siRNA control means KYSE70 cells transfected with the non-specific siRNA. KYSE70 siRNA means KYSE70 cells transfected with the specific siRNA for Notch1. Notch1 siRNA knockdown in the KYSE70 cells significantly increases their 5-FU chemosensitivity both in normoxia (B) and hypoxia (C).

Figure 6. Variable Notch1 and Hes1 expressions revealed in the tissue microarray sections.

Upper panel is for Notch1 expression and lower panel is for Hes-1 expression. All images were taken at 200 x.

Figure 7. Immunohistochemical results of Notch1.

(a) KYSE70 cells with mainly cytoplasmic and membrane staining in most of the cells and a few cells with nuclear staining. (b) a negative Notch1 tumor. (c) normal esophagus epithelial basal cells with cytoplasmic and membrane staining. (d, e and f) represent weak, moderate and strong Notch1 expressions in different squamous cell carcinomas, respectively. In addition, strong Notch1 expression is shown within vascular structures (g) and an infiltration front (h). All images were taken at 200 x.

Figure 8. Immunohistochemical results of Hes1.

(a) positive control of known Hes1 positive breast carcinoma. (b, c and d) represent weak, moderate and strong Hes1 expressions in squamous cell carcinomas, respectively. (e) negative Hes1 in a normal esophagus epithelium. (f) negative Hes-1 in a squamous cell carcinoma. All images were taken at 200 x.

Figure 9. Overall survival curves.

Significantly shorter overall survival (in month) is shown for the patients with higher levels of Notch1 (p<0.001), but Hes-1 expression is not correlated to survival (p = 0.442).