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. 2015 Sep 15;6(27):23720-34.
doi: 10.18632/oncotarget.4690.

Critical role of Wnt/β-catenin signaling in driving epithelial ovarian cancer platinum resistance

Anil Belur Nagaraj  1 , Peronne Joseph  1 , Olga Kovalenko  1 , Sareena Singh  2 , Amy Armstrong  2 , Raymond Redline  3 , Kimberly Resnick  2 , Kristine Zanotti  2 , Steven Waggoner  2 , Analisa DiFeo  1
Affiliations

Critical role of Wnt/β-catenin signaling in driving epithelial ovarian cancer platinum resistance

Anil Belur Nagaraj et al. Oncotarget. .

Abstract

Resistance to platinum-based chemotherapy is the major barrier to treating epithelial ovarian cancer. To improve patient outcomes, it is critical to identify the underlying mechanisms that promote platinum resistance. Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells, but are unable to eliminate cancer initiating cells (CIC). To date, the relevant pathways that regulate ovarian CICs remain elusive. Several correlative studies have shown that Wnt/β-catenin pathway activation is associated with poor outcomes in patients with high-grade serous ovarian cancer (HGSOC). However, the functional relevance of these findings remain to be delineated. We have uncovered that Wnt/β-catenin pathway activation is a critical driver of HGSOC chemotherapy resistance, and targeted inhibition of this pathway, which eliminates CICs, represents a novel and effective treatment for chemoresistant HGSOC. Here we show that Wnt/β-catenin signaling is activated in ovarian CICs, and targeted inhibition of β-catenin potently sensitized cells to cisplatin and decreased CIC tumor sphere formation. Furthermore, the Wnt/β-catenin specific inhibitor iCG-001 potently sensitized cells to cisplatin and decreased stem-cell frequency in platinum resistant cells. Taken together, our data is the first report providing evidence that the Wnt/β-catenin signaling pathway maintains stem-like properties and drug resistance of primary HGSOC PDX derived platinum resistant models, and therapeutic targeting of this pathway with iCG-001/PRI-724, which has been shown to be well tolerated in Phase I trials, may be an effective treatment option.

Keywords: cancer initiating cells; patient derived xenograft; platinum resistance; tumor spheres; β-catenin.

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Conflict of interest statement

CONFLICTS OF INTEREST

None.

Figures

Figure 1
Figure 1. Wnt/β-catenin signaling is up-regulated in platinum-resistant HGSOC tumors
A. Cisplatin treatment study in PDX tumors isolated from platinum-resistant patient (recurred within 6 months, right panel) and platinum sensitive (currently in remission >10 months, left panel). Mice were treated with 2.5mg/kg of cisplatin or PBS control after tumor implants reached ∼120mm3. B. 48h MTT assay with cisplatin confirming platinum resistance of OV145 in-vitro. C. Real-time PCR analysis showing increased mRNA expression of numerous Wnt/β-Catenin target genes in the platinum-resistant OV145 cell line, as compared to OV81 platinum-sensitive cell line. D. TOP-eGFP flow cytometry analysis showing increased TOP-eGFP% in platinum resistant OV145, as compared to platinum sensitive OV81.2.
Figure 2
Figure 2. Generation of primary HGSOC PDX derived platinum-resistant model CP10 to understand mechanisms underlying platinum resistance in HGSOC
A. 48h MTT assay showing 3-fold increase in IC50 value of cisplatin in CP10 platinum-resistant derivative of OV81.2 HGSOC cell line [48μM vs 14.7μM, p<0.0001]. B. Annexin-PI staining showing significantly less cell death in CP10, as compared to OV81.2 upon treatment with 5μM cisplatin for 72hrs. C. Clonogenics cell survival assay showing decreased sensitivity to platinum-therapy in CP10, as compared to OV81.2 upon treatment with 1μM and 2μM cisplatin for 7 days.
Figure 3
Figure 3. Long-term platinum therapy up-regulates the activity of the Wnt/β-catenin pathway in primary HGSOC
A. 10×10 stitch imaging 10x and integrated analysis by METAMORPH software, showing increased tumor sphere formation in platinum-resistant CP10 and CP70 cells, as compared to matched platinum-sensitive OV81.2 and A2780, assessed on day 6. B. Western blots showing increased β-Catenin protein level in CP10 and CP70 platinum-resistant cells, as compared to their sensitive counterparts OV81.2 and A2780 respectively. C. Flow cytometry analysis showing increased TOP-eGFP reporter activity assessed 72hrs after transfection in CP10 and CP70, as compared to OV81.2 and A2780 respectively. D. Correlation analysis showing TOP-eGFP reporter activity is directly proportional to IC50 of Cisplatin in both platinum-sensitive and resistant cells. E. Real-time PCR showing increased mRNA expression of numerous Wnt/β-Catenin target genes in platinum-resistant cells. [* p<0.01, **p<0.001, ***p<0.0001 as compared to controls].
Figure 4
Figure 4. Stable overexpression of β-Catenin confers platinum resistance
A. Western blot showing overexpression of β-CateninS33Y in A2780 and OV81.2. B. Flow cytometry analysis showing increased TOP-eGFP reporter activity, assessed 72hrs after transfection in A2780 βS33Y and OV81.2 βS33Y stable cell lines, as compared to their corresponding controls. C., D. and E. Real-time PCR showing increased mRNA expression of Wnt/β-catenin transcriptional targets upon β-CateninS33Y overexpression. F. 24h MTT assay showing increased cisplatin IC50 in A2780 βS33Y [6.0μM to 9.5μM, p<0.01] and OV81.2 βS33Y [4.0μM to 9.7μM, p<0.001] stable cell lines. G. Annexin-PI staining showing a 2-fold decreased cell death in OV81.2 βS33Y cells, as compared to OV81.2 control cells, upon treatment with 5μM cisplatin for 72hrs (p<0.0001).
Figure 5
Figure 5. β-catenin knockdown induces chemo-sensitivity in platinum-resistant ovarian tumor cells
A. and B. β-Catenin knockdown in platinum-resistant CP70 and CP10 cells, confirmed by β-Catenin [CTNNB1] mRNA down-regulation. A. and protein down-regulation B.. C. Flow cytometry analysis showing decreased TOP-eGFP reporter activity, assessed 72hrs after transfection in CP70 and CP10 sh β-Catenin stable cell lines, as compared to their corresponding shRNA control cell lines. D. 48h MTT assay showing decrease in cisplatin IC50 upon β-Catenin knockdown in platinum-resistant cells CP70 [15.4μM to 7.2μM, p<0.02] and CP10 [∼19.5μM to ∼6.7μM, p<0.01]. E. Clonogenics assay upon 7 days treatment with 1μM and 2 μM cisplatin, showing increased sensitivity to cisplatin upon β-catenin knockdown in both CP70 and CP10. F. Annexin-PI staining upon treatment with 10μM Cisplatin for 72hrs, showing ∼ 6 fold increase in cell death in CP10 upon β-catenin knockdown. G. Real-time PCR showing β-Catenin knockdown significantly decreases mRNA expression of Wnt/β-Catenin signaling-regulated stem cell markers in platinum-resistant CP10 cells. H..Representative 10x light microscopy images and 10×10 stitch imaging 10x and integrated analysis by METAMORPH software showing decreased tumor sphere formation in CP10 upon β-Catenin knockdown. I. Extreme Limiting dilution tumor sphere analysis [ELDA] showing decrease in stem cell frequency in CP10 upon β-Catenin knockdown.
Figure 6
Figure 6. β-Catenin drives platinum resistance in ovarian cancer CICs
A. ALDEFLOUR flow cytometry assay showing increased ALDH activity in platinum-resistant CP70 cells compared to platinum-sensitive A2780 cells. B. 48h MTT assay showing ALDHpos CP70 cells are significantly more resistant to cisplatin (p<0.05). C. Tumor sphere formation is higher in ALDHpos CP70 cells, as assessed by 10×10 stitch imaging 10x and integrated analysis by METAMORPH software. D..β-Catenin knockdown in platinum-resistant ALDHpos CP70 cells confirmed by β-Catenin mRNA and protein down regulation. E..Flow cytometry analysis showing decreased TOP-eGFP reporter activity in ALDHpos CP70 cells stably expressing sh-β-Catenin. F. β-Catenin knockdown decreases mRNA expression of Wnt/β-Catenin-regulated stem cell markers LEF-1, LGR5, CD24, EpCAM and ALDH1A1 in platinum-resistant ALDHpos CP70. G. β-Catenin knockdown in platinum-resistant ALDHpos CP70 cells decreases tumor sphere formation, as assessed by 10×10 stitch imaging 10x and integrated analysis by METAMORPH software. H. β-Catenin knockdown decreases cisplatin IC50 of platinum-resistant ALDHpos CP70 cells, as assessed by 48h MTT assay (p<0.001).
Figure 7
Figure 7. β-Catenin maintains platinum resistance in ovarian cancer CICs
A. FACS sorting of TOP-eGFPlow and TOP-eGFPhigh cells from ALDHpos CP70 cells showing difference in eGFP expression between the sorted populations.B. Real time PCR showing higher expression of Wnt/β-Catenin regulated stem cell markers LEF-1, LGR5, CD24 and EpCAM in TOP-eGFPhigh subpopulationof ALDHpos CP70 cells C. and D. TOP-eGFPhigh subpopulation of ALDHpos CP70 cells are platinum-resistant when compared to TOP-eGFPlow subpopulationof ALDHpos CP70 cells, as assessed by D. 48h MTT assay (p<0.0001), and exhibit increased survival in response to cisplatin treatment, as assessed by E. clonogenics assay on day 7 of cisplatin treatment.
Figure 8
Figure 8. Therapeutic targeting of β-Catenin overcomes platinum resistance
A. iCG-001 decreases TOP-Flash luciferase activity in CP70 and CP10. B. iCG-001 down-regulates Wnt/β-Catenin gene expression in platinum-resistant CP70 and CP10 cells. C. and D. limiting dilution tumor sphere assay with ELDA analysis showing decrease in stem cell frequency in CP10 and CP70 C. and ALDHpos CP70 D. by 5μM iCG-001. E. Isobologram analysis of 48h MTT assay with cisplatin and iCG-001 combination, showing synergistic effect in CP10 (CI=0.8), additive effect in CP70 (CI=1) and ALDHpos CP70 (CI=1).
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References

    1. Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA: a cancer journal for clinicians. 2014;64:9–29. - PubMed
    1. Cooke SL, Brenton JD. Evolution of platinum resistance in high-grade serous ovarian cancer. The Lancet Oncology. 2011;12:1169–1174. - PubMed
    1. Ahmed N, Abubaker K, Findlay J, Quinn M. Cancerous ovarian stem cells: obscure targets for therapy but relevant to chemoresistance. Journal of cellular biochemistry. 2013;114:21–34. - PubMed
    1. Valent P, Bonnet D, De Maria R, Lapidot T, Copland M, Melo JV, Chomienne C, Ishikawa F, Schuringa JJ, Stassi G, Huntly B, Herrmann H, Soulier J, Roesch A, Schuurhuis GJ, Wohrer S, et al. Cancer stem cell definitions and terminology: the devil is in the details. Nature reviews Cancer. 2012;12:767–775. - PubMed
    1. Kim CF, Dirks PB. Cancer and stem cell biology: how tightly intertwined? Cell stem cell. 2008;3:147–150. - PubMed

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