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. 2009 Jan 15;69(2):573-82.
doi: 10.1158/0008-5472.CAN-08-2088.

gamma-Secretase inhibitors abrogate oxaliplatin-induced activation of the Notch-1 signaling pathway in colon cancer cells resulting in enhanced chemosensitivity

Raymond D Meng  1 , Christopher C Shelton, Yue-Ming Li, Li-Xuan Qin, Daniel Notterman, Philip B Paty, Gary K Schwartz
Affiliations

gamma-Secretase inhibitors abrogate oxaliplatin-induced activation of the Notch-1 signaling pathway in colon cancer cells resulting in enhanced chemosensitivity

Raymond D Meng et al. Cancer Res. .

Abstract

Because Notch signaling is implicated in colon cancer tumorigenesis and protects cells from apoptosis by inducing prosurvival targets, it was hypothesized that inhibition of Notch signaling with gamma-secretase inhibitors (GSI) may enhance the chemosensitivity of colon cancer cells. We first show that the Notch-1 receptor, as well as its downstream target Hes-1, is up-regulated with colon cancer progression, similar to other genes involved in chemoresistance. We then report that chemotherapy induces Notch-1, as oxaliplatin, 5-fluorouracil (5-FU), or SN-38 (the active metabolite of irinotecan) induced Notch-1 intracellular domain (NICD) protein and activated Hes-1. Induction of NICD by oxaliplatin was caused by an increase in the activity and expression of gamma-secretase complex, as suppression of the protein subunit nicastrin with small interfering RNA (siRNA) prevented NICD induction after oxaliplatin. Subsequent inhibition of Notch-1 signaling with a sulfonamide GSI (GSI34) prevented the induction of NICD by chemotherapy and blunted Hes-1 activation. Blocking the activation of Notch signaling with GSI34 sensitized cells to chemotherapy and was synergistic with oxaliplatin, 5-FU, and SN-38. This chemosensitization was mediated by Notch-1, as inhibition of Notch-1 with siRNA enhanced chemosensitivity whereas overexpression of NICD increased chemoresistance. Down-regulation of Notch signaling also prevented the induction of prosurvival pathways, most notably phosphoinositide kinase-3/Akt, after oxaliplatin. In summary, colon cancer cells may up-regulate Notch-1 as a protective mechanism in response to chemotherapy. Therefore, combining GSIs with chemotherapy may represent a novel approach for treating metastatic colon cancers by mitigating the development of chemoresistance.

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Figures

Figure 1
Figure 1. Overexpression of Notch-1 in Colon Cancer Progression
A, In a microarray analysis of genes that are overexpressed during colon cancer progression from primary tumors to metastatic liver lesions, we identified several known chemoresistance genes, including BCL2 (bcl-2), BIRC5 (survivin), and CCND1 (cyclin D1), but also a novel target usually involved in developmental signaling, NOTCH1. The expression level of NOTCH1 is enhanced in metastatic lesions (Stage IV and liver metastases), compared to normal colon mucosa or liver parenchyma. In contrast, a related Notch receptor, NOTCH2, is not increased in expression during colon cancer progression. B, HES1, a downstream transcriptional target of Notch-1, is also overexpressed during colon cancer stage progression. C, A positive regulator of Notch-1, GSK3B (GSK-3-beta), which prevents the degradation of the NICD, is enhanced during colon cancer progression, whereas the expression of a negative Notch regulator NUMB, which binds the NICD and prevents its translocation into the nucleus, is suppressed in advanced colon cancers. D, Expression of the Notch-1 ligand JAG1 is not increased during colon cancer progression.
Figure 2
Figure 2. Oxaliplatin Induces Notch-1 Protein and Activity
A, The transcriptionally active Notch-1 intracellular domain (NICD) protein increased with oxaliplatin in a dose-dependent manner in two colon cancer cell lines HCT116 and SW620, with wild-type and mutant p53 respectively (top panel). The two cell lines were treated with increasing doses of oxaliplatin for 48 hours, and total protein was immunoblotted for NICD. Equal protein loading was confirmed by probing for α-tubulin. Next, using a luciferase reporter containing the promoter of the Notch-1 target HES-1. we report that oxaliplatin also induces Hes-1 transcriptional activity in a dose-dependent manner (bottom panel) in both colon cancer cell lines. Cells were first transfected with the Hes-1 luciferase construct and then total protein was harvested. All values were normalized for transfection efficiency and are expressed as a percentage of the untreated controls ± SEM B, Hes-1 luciferase activity was assayed in a panel of colon cancer cell lines following treatment with oxaliplatin (1 or 2 μM) for 48 hours. A statistically significant dose-dependent increase in Hes-1 luciferase activity was observed in each cell line, although the degree of induction varied. C, In HCT116 cells, the induction of NICD protein by oxaliplatin also increases the expression of downstream targets of Notch-1, including Hes1, cyclin D1, and survivin. Equal protein loading was demonstrated by probing for α-tubulin protein expression. D, Both the chemotherapeutic antimetabolite fluorouracil (5-FU) and the topoisomerase I inhibitor irinotecan, metabolized to SN-38, increase the transcriptional activity of a Hes-1 luciferase reporter in HCT116 cells in a dose-dependent manner. All values were normalized for transfection efficiency and are expressed as a percentage of the untreated controls ± SEM.
Figure 3
Figure 3. Oxaliplatin Induces Gamma-Secretase Protein Expression
A, In HCT116 colon cancer cells, oxaliplatin treatment (0.5 μM) for 48 hours induces the protein expression of two subunits of the gamma-secretase protein complex, nicastrin and presenilin-1. Equal protein loading was confirmed by probing for α-tubulin expression. B, siRNA (60 pmol/L) to the nicastrin protein subunit of the gamma-secretase complex suppresses nicastrin protein expression at 48 hours (top panel), which subsequently decreases the activity of a Hes-1 luciferase reporter in HCT116 cells (bottom panel). All values were normalized for transfection efficiency and are expressed as a percentage of the untreated controls ± SEM. C, HCT116 cells were transfected with siRNA to nicastrin (60 pmol/L) for 24 hours and then treated with oxaliplatin (1 μM) for an additional 24 hours. siRNA to nicastrin not only abrogates the induction of nicastrin protein previously observed with oxaliplatin treatment, but also suppresses the increase in NICD and cyclin D1 protein usually observed after oxaliplatin. Equal protein loading was demonstrated α-tubulin protein expression.
Figure 4
Figure 4. Gamma-Secretase Inhibitors Abrogate Notch-1 Induction by Oxaliplatin
A, HCT116 colon cancer cells were co-treated with increasing doses of oxaliplatin and either the gamma-secretase inhibitor (GSI) GSI34 at 10 μM (indicated by “+” signs) or 0.1% DMSO for 48 hours (indicated by “-“ signs). Whereas oxaliplatin induced NICD expression in a dose-dependent manner, co-treatment with GSI34 abrogated this induction at each dose. Correspondingly, GSI34 (bottom panel, black bars) also abrogated the induction of Hes-1 luciferase activity by oxaliplatin at both dose levels (bottom panel, light gray bars). B, A panel of colon cancer cell lines was treated with oxaliplatin (1 μM) combined with either GSI34 (10 μM) or 0.1% DMSO for 48 hours to assess Hes-1 activity. GSI34 (black bars) abrogated the induction of Hes-1 by oxaliplatin in each colon cancer cell line, although the effect lessened in cell lines with high induction of Hes-1, such as SW620. C, HCT116 were treated with 0.1% DMSO control, oxaliplatin (1 μM), GSI34 (10 μM), or the combination of both drugs for 48 hours, and colonies were counted after 14 days. Although GSI34 and oxaliplatin both decreased colony formation, the combination of both drugs produced the most significant suppression of viability (Student's t-test, p=0.034). Similarly, the combination of GSI34 with either 5-FU (8 μM) or SN-38 (20 nM) also suppressed colony formation more than either treatment alone. All values are expressed as a percentage of colonies formed in the DMSO controls ± SEM. D, HCT116 cells were co-treated with oxaliplatin (0.5 μM) and with either GSI34 (10 μM) or 0.1% DMSO for 48 hours and stained with 4'-6-diamidino-2-phenylindole (DAPI). As visualized by confocal immunofluorescence microscopy, the number of cells with fragmented nuclei, suggestive of apoptotic cells, were increased by the combination treatment (p<0.05). Values are expressed as the percentage of cells with nuclear fragmentation ± SEM.
Figure 5
Figure 5. Notch-1 Expression Affects Chemosensitivity
A, HCT116 cells were transfected with siRNA (60 pmol/L) to Notch-1 or to control random sequences for 48 hours. Notch-1 siRNA decreases both the protein expression of Notch-1 (top panel) and the activity of a Hes-1 luciferase reporter (bottom panel). HCT116 cells were then transfected with siRNA to Notch-1 or to control sequences (as indicated) for 12 hours and then treated with either oxaliplatin (0.5 μM) or media for an additional 48 hours. The combination of Notch-1 siRNA and oxaliplatin treatment decreased colony formation more than either treatment alone (Student's t-test, p=0.011). B, HCT116 cells were transfected with pCS2-NICD (1 μg) for 24 hours, and NICD expression increases in a dose-dependent manner (top panel). NICD overexpression also enhances Hes-1 luciferase activity after 48 hours (bottom panel). HCT116 cells were then transfected with pCS2-NICD (2 μg) or pCS2 empty vector for 12 hours and were treated with oxaliplatin (0.5 μM) or media alone for 48 hours. Whereas vector-transfected cells showed a decrease in colony formation, expression of NICD protected the cells from oxaliplatin (p<0.05). C, HCT116 cells were transfected with siRNA to Notch-1 or to control sequence (both at 60 pmol/L), and were then treated with either SN-38 (0.2 nM) for 48 more hours. The combination of Notch-1 siRNA and SN-38 decreased viability significantly, compared to either treatment alone (p=0.022) as measured by the sulforhodamine B assay. In contrast, overexpression of NICD following transfection of pCS2-NICD (1 μg) in HCT116 cells increased chemoresistance to SN-38 (0.2 nM).
Figure 6
Figure 6. Gamma-secretase inhibitors decrease the induction of pro-survival factors, including Akt signaling, by oxaliplatin
A, HCT116 cells were treated with both oxaliplatin and either GSI34 (10 μM) or 0.1% DMSO for 48 hours before total protein lysates were probed with antibodies, as indicated. GSI34 decreased the expression of Bcl-XL and survivin following oxaliplatin and blunted the increases in cyclin D1 after oxaliplatin (top panels). Co-treatment with GSI34 also decreased Phospho-Akt-Ser473, mTOR, and Phospho-S6Ser235/Ser236 proteins following oxaliplatlin (bottom panels). Total S6 and DNA-PK protein expression were also decreased by co-treatment with GSI34 but not by oxaliplatin. Equal protein loading was demonstrated by probing for tubulin expression. B, HCT116 cells were transfected with siRNA targeting Akt (50 nM) for 48 hours, and total Akt protein levels were decreased (top panel). The percentage of colonies formed was also decreased by siRNA to Akt following oxaliplatin treatment (bottom panel), compared to cells transfected with control siRNA (p<0.05). C, Following treatment with chemotherapy (oxaliplatin, 5-FU, or SN-38), colon cancer cells activate the Notch pathway by upregulating the gamma-secretase protein complex through induction of the nicastrin (NCT) and presenilin-1 (PS1) subunits to cleave more NICD (top panel). The NICD proteins then activate targets involved in chemoresistance, such as PI3K/Akt, Bcl-XL, survivin, and cyclin D1. In contrast, downregulation of Notch signaling chemosensitizes cells as pro-survival targets are no longer activated (bottom panel). The Notch pathway can be suppressed if the colon cancer cells are co-treated with GSIs, which prevent the formation of the cleaved NICD by blocking the gamma-secretase protein complex. Therefore, the colon cancer cell is more chemosensitive after inhibition of Notch signaling.
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