Dissecting the heterogeneity of cancer stem cells in a single cell-derived tumor: implications in anti-cancer drug development
A tumor is a hierarchically organized tissue with a distinct fraction of tumorigenic cells (CSCs) generating the bulk of the non-tumorigenic cells (Shackleton M, et al, 2009). Tumor heterogeneity is believed to be the main contributor to drug resistance and tumor recurrence by initiating phenotypic and functional diversities in tumor cells (Yap TA et al, 2012). Therefore, targeting these locomotive drivers of tumorigenesis, the CSCs, should be a high priority in pursuit of more effective cancer therapies (Zhou BB, et al, 2009). One of the fundamental characteristics of the CSCs is self-renewal, that is cell division with maintenance of the stemness state. Self-renewal in most CSCs is maintained by the same signaling pathways as in normal stem cells, such as Wnt/ß-catenin signaling pathway (Zhou BB, et al, 2009, Takebe N, et al, 2011). Wnt/ß-catenin pathway is frequently dysregulated in many types of cancers and specifically within subpopulations with stem-like properties (Li L, et al, 2010, Takebe N, et al, 2011). Blocking Wnt signaling is a very attractive strategy since such developmental pathway is not generally required for normal differentiated adult cells. During past years, we have attempted to discover Wnt inhibitors by screening clinically approved drugs with well characterized bioactivity and safety profiles. Axitinib, a receptor tyrosine kinase inhibitor approved for treatment of renal cell carcinoma, showed strong inhibition of Wnt signaling activated by GSK3 inhibitor 6-bromoindirubin-3’-oxime (6BIO) and ß-catenin lacking the N-terminal in culture cells. In addition, Axitinib inhibited Wnt signaling and promoted ß-catenin turnover in SW480 cancer cells harboring APC with mutation commonly detected in cancer patients, supporting that Axitinib blocks the Wnt/ß-catenin signaling downstream and independent of the GSK3ß/APC destruction complex. We also evaluated the Wnt inhibition in zebrafish models. In fish embryos Axitinib dose dependently rescued the eyeless phenotype induced by abnormal activation of Wnt/ß-catenin signaling, and strongly inhibited the tailfin regeneration and Wnt signaling activation in adult TCF-GFP transgenic fish, directly demonstrating that Axitinib inhibits Wnt/ß-catenin signaling in vitro and in vivo. We further identified a number of candidate drug binding proteins using chemoproteomics approachs DARTS/DIGE/MS DARTS (drug affinity-responsive target stability assay coupled to two dimensional difference in gel electrophoresis and mass spectrometry) and MST (microscale thermophoresis) assay. Notably, in drug treated cancer cells we have observed asymmetric division in terms of Wnt signaling, ?-catenin and DNA segregation that is a well-established ACD feature of stem cells. Correlation analysis revealed that Axitinib induced non-random DNA segregation is dependent on the ß-catenin degradation. Given that loss of ACD is critical for tumor initiation and progression, re-establishment of ACD in cancer cells could be a mechanism of tumor suppression and an opportunity to selectively target tumor cells with less effect on adult tissue homeostasis. In addition the publications in Proc Natl Acad Sci U S A 2016; 113:9339-44; PMID:27482107 and Cell Cycle 2016; PMID: 27687486, DOI: 10.1080/15384101.2016.1235850 and a manuscript under 2nd revision by Nature Chemical Biology ((NCHEMBA160704154A), with the assistance by Bergen Technology Transfer (BTO), our findings have been filed for patent ( “ Newly identified Wnt/beta -catenin signal transduction inhibitors and the use thereof in the treatment or prevention of diseases and conditions in which Wnt/beta-catenin signal transduction is a contributing factor.Filed 07 Dec 2016, the United Kingdom, International (PCT) Patent Application No. PCT/EP2016/076171).
Cancer is a leading cause of human death, and most advanced solid tumors remain incurable. Large scale tumor sequencing has identified Wnt/ß-catenin signaling is one of most frequently mutated pathways in major cancer types, however, drugs targeting Wnt/ß-catenin signaling pathway is lacking for clinical use. In this project, we have identified Axitinib, a receptor tyrosine kinase inhibitor approved for clinical treatment of renal cell carcinoma, showed strong inhibition of Wnt/ß-catenin signaling in vitro and in vivo. The work opens for further investigation of the regulatory principles and to verify whether chemical derivatives of Axitinib may have a greater inhibition to Wnt signaling driven tumor growth. In addition, this work supports a recognized strategy where a drug which is already approved for human use, is potentially effective to new targets. We now understand that small molecule substances might have more than one molecular target in cells. This strategy, called repurposing, is also potentially fruitful because we already know that these drugs are introduced to and distributed in the organism with acceptable and known side effects. The publication in PNAS was selected to be highlighted in the Editors' Choice section of Science Signaling, the weekly journal from the publisher of Science magazine, and has been selected for an Editorial feature published in the journal Cell Cycle.
Identification of novel inhibitors of Wnt/ß-catenin signaling
During the last year, we have finished a revised manuscript. In addition, we have identified the direct target of another Wnt/ß-catenin signaling inhibitor, and the significant correlation between the target protein and the survival periods of patients with major human cancer types, highlighting the potential of the inhibitors as cancer therapeutic agents.
Wnt/ß-catenin signaling is frequently mutated in major cancer types. In this project, we have discovered 5 novel Wnt inhibitors and identified direct targets of two inhibitors. Currently, the manuscript titled "small molecule targets Wnt/ß-catenin signaling and directs asymmetric cell division in cancer" is reviewed by Nature Communications, in which we have demonstrated Axitinib targeting Wnt/ß-catenin signaling in vitro and in vivo, the identification of direct target proteins suggests that Axitinib targets the endpoint of pathway. In addition, a patent application based on these findings has been registered together with Bergen Teknologioverføring. Recently, we have identified the direct target of another compound 1B8, a clinically approved anti-parasitic drug that efficiently inhibits oncogenic Wnt/ß-catenin by promoting ß-catenin degradation independent of GSK3ß and APC. Treatment with 1B8 stabilizes the target protein thus increasing the turnover of ß-catenin in cancer cells and tumors. Examination of human cancer patient samples revealed significant decreased expression the target protein in major cancer types, including colorectal cancer, prostate cancer, kidney cancer and melanoma. Examination of the large cancer genomic datasets revealed high correlation between the target protein and the Wnt/ß-catenin signaling markers and patient survival periods, Overexpression of the target protein inhibits the growth of APC or CTNNB1 mutant colon cancer cells and organoids, supporting the Wnt inhibition and tumor suppression by the target protein in cancer and highlighting the potential of 1B8 as a promising therapeutic agent for cancer patients with Wnt signaling mutations. The manuscript is ready to be submitted to an international high impact journals.
Indentification of novel inhibitors to Wnt/ß-catenin signaling
During the last year, we have successfully identified 5 novel inhibitors of Wnt signaling and their direct target proteins. These compounds showed effective inhibition to Wnt dependent tumor progression in mice strongly indicating the high potential to clinical treatment of cancer patients with Wnt pathway mutations.
Wnt/ ß-catenin signaling is one of the most attractive therapeutic targets of cancer especially tumor initiating cells. In this project, we have identified 5 novel Wnt inhibitors targeting the endpoint of the pathway. We have demonstrated the inhibition to 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 to 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 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 together with Bergen Teknologioverføring.
Dissecting the heterogeneity in a single cell-derived tumor
Tumor heterogeneity is believed to be the main contributor to drug resistance and tumor recurrence, a common cause of poor clinical outcome of cancer. This project is designed to dissect the heterogeneity of cancer stem cells (CSCs) in a single cell-derived tumor model and develop drugs targeting all the CSCs to achieve complete cures of tumor.
During the last years, we have established a single cell-derived tumor model and representing the first tumorigenic prostate model without the use of external oncogenes (Yi Qu, at el, 2013). Importantly, we have found that generation of prostate tumor-initiating cells (EPT3) is associated with elevation of reactive oxygen species and IL-6/STAT3 signaling.
Accumulated evidences have demonstrated a significant role of WNT/ß-catenin signaling in cancer stem cells or tumor initiating cells.In this study, we have detected and isolated a small minority of EPT3 cells with high Wnt signaling activity using a fluorescence-based Wnt pathway reporter. indicating there are distinct cell populations with different signaling pathway activities in a single cell derived tumor.
In a pilot screening of Wnt inhibitors, 5 out of 500 compounds have been found inhibiting Wnt signaling pathway in both Topflash reporter assay and Top-fluorescent reporter assay. The inhibition of Wnt signaling by these compounds were further supported by the repressed expression of Wnt target genes in compounds treated prostate cancer EPT3 cells in Western blotting assay and quantitative reverse transcription PCR. Additionally, all these compounds inhibited Wnt signaling-involved developmental phenotypes in zebrafish embryos.
The plans in the next year will be 1) identification of the tumor initiation of the.Wnt (+) cells,2) funcational evaluation of Wnt inhibition by these candidate compounds, 3) examination of the anti-cancer ability of these compounds in vitro and in vivo.
Small molecule induces Wnt asymmetry in cancer.
Cell Cycle 2016 Sep 29. Epub 2016 sep 29
Axitinib blocks Wnt/ß-catenin signaling and directs asymmetric cell division in cancer.
Proc Natl Acad Sci U S A 2016 Aug 16;113(33):9339-44. Epub 2016 aug 1
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
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