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. 2013 Aug 6;109(3):667-75.
doi: 10.1038/bjc.2013.361. Epub 2013 Jul 18.

Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a γ-secretase inhibitor, in a preclinical colorectal explant model

J J Arcaroli  1 , K S Quackenbush, A Purkey, R W Powell, T M Pitts, S Bagby, A C Tan, B Cross, K McPhillips, E-K Song, W M Tai, R A Winn, K Bikkavilli, M Vanscoyk, S G Eckhardt, W A Messersmith
Affiliations

Tumours with elevated levels of the Notch and Wnt pathways exhibit efficacy to PF-03084014, a γ-secretase inhibitor, in a preclinical colorectal explant model

J J Arcaroli et al. Br J Cancer. .

Abstract

Background: Dysregulation of the Notch pathway has been identified to play an important role in the development and progression of colorectal cancer (CRC). In this study, we used a patient-derived CRC explant model to investigate the efficacy of the clinical γ-secretase inhibitor (GSI) PF-03084014.

Methods: A total of 16 CRC explants were treated with PF-03084014. Knockdown of RBPjκ gene was used to determine the specificity of PF-03084014. Evaluation of the Notch and Wnt pathways in CRC explant tumours was performed by gene array and immunoblotting.

Results: We identified a subset of CRC tumours that exhibited elevations of the Notch and Wnt pathways sensitive to PF-03084014. Treatment with the GSI resulted in a significant reduction in cleaved Notch, Axin2 (Wnt-dependent gene) and active β-catenin. In addition, knockdown of the RBPjκ gene showed that PF-03084014 has specificity for the Notch pathway in an HCT116 cell line xenograft model. Finally, an increase in apoptosis was observed in CRC001- and CRC021-sensitive tumours.

Conclusion: This study provides evidence that inhibition of γ-secretase may be beneficial in a subset of patients with elevated levels of the Wnt and Notch pathways.

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Figures

Figure 1
Figure 1
PF-03084014 effects on tumour growth in CRC explants. (A) Sixteen CRC explants were treated with PF-03084014 125 mg kg−1 per day BID by oral gavage for 28 days. Tumour size was evaluated twice per week by caliper measurements using the formula: tumour volume=(length × width2) × 0.52. Tumour growth index (TGI) was calculated by relative tumour growth of treated mice divided by relative tumour growth of control mice × 100. Cases with a TGI of <50% were considered as sensitive, and TGI of >50% were considered as resistant to PF-03084014. Three xenografts (CRC001, CRC021, and CRC040) were sensitive to PF-03084014 (TGI⩽50%) and thirteen xenografts were resistant to PF-03084014 (TGI>50%). Columns, mean (n=8–10 tumours per group); bars, s.e. *, significance (*P<0.05), (***P<0.001) compared with vehicle-treated tumours. (B) A representative figure of a sensitive explant CRC021 and (C) resistant explant CRC042.
Figure 2
Figure 2
Investigation of PF-03084014 as a specific inhibitor of the Notch pathway. (A) Evaluation of RBPjκ knockdown before injection and (B) after treatment of tumours in a xenograft model showed a 75% and 76% knockdown of RBPjκ, respectively. (C) RBPjκ knockdown reduced cleaved Notch1 activity and Hes-1 in xenograft tumours compared with scramble control. PF-03084014 treatment effects on HCT116 scramble control (D) and HCT116 RBPjκ knockdown (E) were evaluated in an in vivo xenograft model. Treatment significantly decreased the growth of HCT116 parental (data not shown) and scramble groups (D). In contrast, PF-03084014 did not have any additional anti-proliferative effects in the RBPjκ knockdown group (E). Knockdown of RBPjκ significantly decreased growth of the HCT116 cell line when compared with HCT116 scramble control (F). Columns, mean (n=8–10 tumours per group); bars, s.e. *, significance (*P<0.05).
Figure 3
Figure 3
Notch pathway analysis between sensitive (CRC040, 021, and 001) vs resistant tumours (CRC020, 007, and 034). (A) KEGG pathway analysis of the Notch pathway shows an increase in many components of the Notch pathway in sensitive tumours when compared with resistant tumours. Red indicates elevated gene expression. (B) Baseline levels of cleaved Notch1 are elevated in sensitive tumours compared with resistant tumours. (C) Densitometry of cleaved Notch1/Actin ratio showed a significant increase in sensitive tumours compared with resistant tumours (P<0.02). (D) PF-03084014 treatment decreased cleaved Notch activity in the sensitive tumour CRC021 (n=3 mice/tumour per group for control and GSI treated). Examination of cleaved Notch by densitometry showed a significant (*P<0.05) decrease with PF-03084014 treatment. (E) No treatment effects were observed in the resistant tumour CRC012.
Figure 4
Figure 4
Wnt pathway analysis between sensitive vs resistant tumours. (A) KEGG pathway analysis of the Wnt pathway shows an increase in many components of the canonical Wnt pathway in sensitive tumours when compared with resistant tumours. Red indicates elevated gene expression. (B) Baseline levels of active β-catenin are elevated in sensitive tumours compared with resistant tumours. (C) Densitometry of active β-catenin/β-catenin ratio showed a significant increase in sensitive tumours compared with resistant tumours (P<0.05). (D) PF-03084014 treatment decreased active β-catenin and Axin2 levels in the sensitive tumour CRC021. (E) No treatment effects were observed in the resistant tumour CRC012.
Figure 5
Figure 5
Assessment of PF-03084014 on apoptosis. (A) Post gene array 3 days after PF-03084014 treatment on gene expression of apoptotic genes in CRC001-sensitive explant. (B) Western blot analysis of cleaved Notch1, cleaved caspase 3, cleaved PARP, phos p65, and BCLxL in CRC021 2 h, 8 h, 24 h and 8 days after treatment with PF-03084014.
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