Screening of Natural Compounds Inhibiting Wnt/ß-catenin Activity in Prostate Cancer Stem Cells
Wnt/ ß-catenin signaling is one of the most attractive therapeutic targets of cancer especially in tumor initiating cells. In this project, we have identified 5 novel Wnt inhibitors targeting the endpoint of the Wnt pathway. We have demonstrated the inhibition of Wnt/ß-catenin signaling by these compounds in various prostate and colon cell lines using Topflash reporter assay, Top-fluorescent reporter assay, realtime PCR and Western blotting. These compounds inhibited the embryonic development and fin regeneration in transgenic TCF-GFP zebrafish, supporting the inhibition of Wnt signaling in vivo. Particularly, these compounds depleted cancer cells with constitutive Wnt signaling by inducing apoptosis and repressing the Wnt activity. In APCmin-/+ mice that spontaneously generated tumors due to a mutation in APC gene and aberrant Wnt signaling, these compounds significantly blocked the tumor progression, strongly supporting the capacity of these compounds in blocking the Wnt signaling dependent tumor growth. Using chemoproteomic approaches, we have found a number of direct binding proteins of these compounds and identified a mediator complex as the target in blocking Wnt signaling. Our findings discovered novel therapeutic agents targeting Wnt signaling and provided a possibility for treatment of cancer patients with genetic mutation that is potential to activate Wnt signaling. Currently, two manuscripts are ready to be submitted to international top journals and two compounds are on the way to patents application in collaboration with Bergen Teknologioverføring.
Conventional anti-cancer drug discovery programs typically select compounds cytotoxic in cancer cell lines versus non-cancer cells. It is logical but has been largely disappointing with regard to registration of new anti-cancer drugs. The cancer stem cells (CSCs) concept provides a new opportunity considering CSCs are indispensable and essential for tumor growth and cancer drug resistance. Wnt/ ß-catenin signaling is critical for the cancer stem cells to maintain the self-renewal and tumor initiating ability, therefore this is one of the most attractive therapeutic targets of cancer stem cells. In addition, cancer genome sequencing studies revealed high frequency of Wnt signaling mutations in various tumors, including APC, CTNNB1, TCF3 (TCF7L1) and TCF4 (TCF7L2) in colorectal tumor, CTNNB1 and AXIN1 in hepatocellular carcinoma (HCC), CTNNB1 in medulloblastoma and endometrial carcinoma, and APC in prostate cancer. Notably, most of these WNT pathway mutations in cancer are predicted to result in aberrant activation of WNT/ß-catenin signaling. In this project, we have successfully identified 5 novel inhibitors of Wnt signaling and their direct target proteins. The effective inhibition of Wnt dependent tumor progression in mice indicates a strong potential to treatment of cancer patients with Wnt pathway mutations.
Characterization of novel Wnt inhibitors
A growing body of evidence illustrates critical roles of Wnt/ß-catenin signaling in cancer stem cells (CSCs) that must be eradicated to achieve cures. Targeting the Wnt signaling is a high priority in development of novel anti-cancer drugs. We have identified several compounds that efficiently and selectively target the Wnt signaling pathway.
During the last years, we have established a prostate tumor-initiating cell model that is very attractive in discovery and development of anti-cancer drugs. In addition, we have identified several leading compounds that efficiently and selectively target the Wnt signaling pathway.
Targeting of Wnt signaling by our candidate compounds has been demonstrated in many assays. All these compounds inhibited Wnt signaling pathway in both Topflash reporter assay and Top-fluorescent reporter assay. The repressed expression of Wnt target genes Axin2, Lef1 and Tcf7 in compounds treated prostate cancer EPT3M1 cells has been evidenced by Western blotting assay and quantitative reverse transcription PCR. Additionally, all these compounds showed high (2 compounds) or partial (3 compounds) inhibition of Wnt signaling-involved developmental process in zebrafish embryos. Finally, our candidate compounds inhibited colony formation of human cancer cell lines SW480 cells and HCT116 cells but not RKO cells, demonstrating the selective targeting of cells with aberrant Wnt signaling activity.
To understand the potential mechanism of our leading compounds in targeting Wnt signaling, we have detected the ß-catenin level upon drug treatment using anti-active-ß-Catenin (anti-ABC) antibody. We found 3 compounds significantly reduced the active-ß-catenin (ABC) level in drug treated SW480 cells, indicating these drugs regulated the process of stabling of ß-catenin. On the other hand, 2 compounds highly blocked the activated Wnt signaling by overexpression of mutated ß-catenin that is resistant to ß-catenin degradation complex, indicating these compounds targeting the Wnt signaling at the nuclear level.
The plans for the next year include the confirmation of the Wnt inhibition in mice and further understand the exact targeting mechanism of all our leading compounds.
Screening inhibitors of Wnt/beta-catenin signaling pathway
Current anti-cancer drug therapies all too often fail due to drug resistance. Many first-line drugs are not effective against cancer stem cells (CSCs) that must be eradicated to achieve cures. Therefore, targeting these drivers of tumorigenesis, the CSCs, should be a high priority in pursuit of more effective cancer therapies.
During the last years, we have established a stepwise prostate carcinogenesis model (EPT3) and representing the first tumorigenic prostate model without the use of external oncogenes. The unique prostate cancer model provides an excellent platform for discovery and development of anti-cancer drugs.
Accumulated evidences have demonstrated a significant role of WNT/ß-catenin signaling in prostate cancer. Using a fluorescence-based Wnt reporter, we have detected and isolated a small minority EPT3 cells with high WNT/ß-catenin signaling activity. Several assays provided evidence that these Wnt (+) cells are putative cancer stem cells.
By collaboration with Prof. Weidong Zhang at the Second Military Medical University, China, we have gotten access to a library containing over 6000 natural compounds purified from 8000 Chinese herbal medicines’ extracts belonging to 342 plant families. In a pilot screening of Wnt inhibitors, 9 out of 600 compounds showed selective cytotoxicity to Wnt(+) cells compared to Wnt(-) cells at 5µM. Very interestingly, 5 of them were purified from the same plant Inula japonica, thus representing a promising strategy to develop anti-cancer drugs using our CSC model and the natural compound library.
The next plans will be to 1) expand the primary screening scale to get more leading compounds, 2) Characterize the the leading compound candidates including pathway specificity and cell line selectivity. 3) Functional evaluation of the leading compounds.
Generation of prostate tumor-initiating cells is associated with elevation of reactive oxygen species and IL-6/STAT3 signaling.
Cancer Res 2013 Dec 1;73(23):7090-100. Epub 2013 okt 7
MiR-182 and miR-203 induce mesenchymal to epithelial transition and self-sufficiency of growth signals via repressing SNAI2 in prostate cells.
Int J Cancer 2013 Aug 1;133(3):544-55. Epub 2013 feb 27