Sensitization of cancer stem cells to chemotherapy through derepression of epigenetically silenced microRNA-203

Keighley Reisenauer, Collin Campion, Joseph TaubeDepartment of Biology, Baylor University

References1. Dent, R., Trudeau, M., Pritchard, K.I., Hanna, W.M., Kahn, H.K., Sawka, C.A., Lickley, L.A., Rawlinson, E., Sun, P., and Narod, S.A. (2007). Triple-Negative

Breast Cancer: Clinical Features and Patterns of Recurrence. Clin Cancer Res 13, 4429–4434.2. Prat, A., Lluch, A., Albanell, J., Barry, W.T., Fan, C., Chacón, J.I., Parker, J.S., Calvo, L., Plazaola, A., Arcusa, A., et al. (2014). Predicting response and survival in

chemotherapy-treated triple-negative breast cancer. Br J Cancer 111, 1532–1541.3. Cancer Genome Atlas. (2012). Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70.4. Liedtke, C., Mazouni, C., Hess, K.R., André, F., Tordai, A., Mejia, J.A., Symmans, W.F., Gonzalez-Angulo, A.M., Hennessy, B., Green, M., et al. (2008). Response

to Neoadjuvant Therapy and Long-Term Survival in Patients With Triple-Negative Breast Cancer. JCO 26, 1275–1281.5. Mani, S.A., Guo, W., Liao, M.-J., Eaton, E.N., Ayyanan, A., Zhou, A.Y., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., et al. (2008). The Epithelial-

Mesenchymal Transition Generates Cells with Properties of Stem Cells. Cell 133, 704–715.

6. Perou, C.M. (2011). Molecular Stratification of Triple-Negative Breast Cancers. The Oncologist 16, 61–70.7. Taube, J.H., Malouf, G.G., Lu, E., Sphyris, N., Vijay, V., Ramachandran, P.P., Ueno, K.R., Gaur, S., Nicoloso, M.S., Rossi, S., et al. (2013). Epigenetic

silencing of microRNA-203 is required for EMT and cancer stem cell properties. Scientific Reports 3.

AcknowledgementsMany thanks to the Taube Lab members for their work in data collection and enthusiastic

support.

Differential sensitivity to targeted agents

Figure 2:

Immortalized human mammaryepithelial cells (HMLE) and HMLEwith a Twist knockout provideideal models to demonstrate thedifferences between epithelialand mesenchymal breast cancersubtypes, respectively. Theseresults simulate the effects ofCSC-targeted drugs on pre- andpost-EMT tumor cells. HMLETwist cells are modestly moresensitive to treatment withdecitabine and vorinostat while,HMLE cells are more sensitive toselumetinib.

Hypothesis: miR-203 repression is necessary for acquisition of CSC properties and compounds that

interfere with miR-203 suppression have potential as anti-CSC targeted

therapies

IntroductionTriple negative breast cancer (TNBC) patients, unlike those withER-, PR-, or HER2-positive breast cancers, face higher rates ofrelapse and mortality, primarily due to the lack of targetedtherapies1-3. While cytotoxic neo-adjuvant chemotherapiesprove effective in 70% of patients, the remaining often developrecurrence and/or distant metastases3,4.

Cancers of epithelial origin grow as tightly compacted tumors,bounded by a basement membrane. In order for metastasis tooccur, tumor cells must lose contact with their neighbors,migrate through the extracellular matrix, enter the vasculature,and survive in circulation in a process called epithelial-mesenchymal transition (EMT). In the reverse process,mesenchymal-epithelial transition (MET) cells exit thevasculature and re-initiate proliferation at a new site to formmetastases.

Tam and Weinberg; Nature Medicine 2014

TNBCs, while having diverse subtype expression, share severalcommonalities that chemotherapeutic and xenobioticcompounds target. These basal-like tumors show a highfrequency of TP53 mutations and EGFR activity. The PI3Kpathway shows increased activity, as well as the RAS-RAF-METpathway3,6.

Dean et al., Nat. Rev. Cancer, 2005

Found in these tumors are cancer stem cells (CSCs), which arepervasive and resistant to chemotherapies. The acquisition ofCSC properties may be linked to EMT by differentiated tumorcells5. Targeting CSCs can help block tumor progression.

Compound Mechanism

Crizotinib ALK/MET inhibitor

Selumetinib MEK-1/ MEK-2 inhibitor

Vorinostat HDAC 1 & 3 inhibitor

Decitabine Hypomethylation agent

Figure 3: Future DirectionSignaling Pathways Necessary for CSCs

miR-203 repression is required for tumor initiation

Voso, et. al. 2014. Haemotologica. EHA.

Figure 1: DNA methylation of MIR203 enablesacquisition of CSC properties

A-D) miR-203 is repressed in CSC-rich fractionsof breast cancer cells and restoration of itsexpression reduces non-adherent growth invitro and tumor initiation in vivo7.

E-G) A high-throughput screen was conductedfor compounds that elevate expression of a miR-203 silenced reporter construct in SUM159cells. Positive controls included the inhibitors ofDNA methylation: 5-azaC and decitabine

H) Five compounds from this screen wereverified to elevate miR-203 expression in MDA-MB-231 cells.

Solomon, B. 2015. MET. My Cancer

Genome

Crizotinib

Selumetinib

Chemotherapeutic drugs target CSCs through avariety of epigenetic and cancer signaling pathways.Crizotinib and Selumetinib act to block componentsof the ALK-RAS pathway. This pathway is involved inthe proliferation and survival of cells and oftentimesshows amplification in tumor populations. Vorinostatfunctions as an HDAC inhibitor by binding to thecatalytic domain of HDAC 1 and 3. This leads to anaccumulation of hyperacetylated histones andtranscription factors, resulting in G1 and G2 arrest.Decitabine inhibits transcription by disrupting DNAmethyltransferase activity.

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Shown to the left is a double stain of HMLE cells from fluorescence-activated cellsorting (FACS) used in flow cytometry depicting distinct cancer subtypes. Circled inred is a subpopulation with cell markers CD44hi/CD24lo, which is consistent withphenotypes of CSCs. Circled in blue is a CD44lo/CD24hi subpopulation that matchesepithelial tumor cells.

Applying these studied compounds at doses less than their IC50 values is expectedresult in differential sensitivities between the CSCs and epithelial tumorsubpopulations. Using miR-203 to repress tumor initiation, in combination withdrug therapies, is expected to yield clinically applicable reduction in metastatic.

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