|A Long Awaited Magic Bullet for Cancer? - “Identification of Drugs Including a Dopamine Receptor Antagonist that Selectively Target Cancer Stem Cells”|
Cancer stem cells (CSCs) are now becoming acknowledged as an important factor in tumor initiation and sustainment (Dick, 2009 and Reya et al) and, unfortunately, it has been shown that conventional chemotherapeutics are often ineffective against human CSCs (Guan et al) and have detrimental effects on healthy stem and progenitor cells (Smith et al). This therefore suggests that residual CSCs can lead to the relapse of disease after long periods of remission and also that current regimes of chemotherapeutics are not well suited to the long term treatment of tumourigenesis and can affect normal cells so that they actually foster tumourigenesis as exemplified by a recent study (Sun et al). Therefore the development of new chemotherapeutics which efficiently target CSCs alone would be highly advantageous. Now, researchers from the group of Mickie Bhatia from McMaster University, Canada, using high content screening of neoplastic and normal human pluripotent stem cells (hPSCs), have discovered that thioridazine, an antipsychotic drug, can selectively target neoplastic cells via their dopamine receptors, while leaving normal cells unaffected (Sachlos et al).
First, the study used a previously described variant hPSC line to mimic the neoplastic properties of somatic CSCs, which importantly included the aberrant block in differentiation capacity (Werbowetski-Ogilvie et al), for high content compound screening in vitro. A reduction in OCT4 and SOX2 levels was used as an indicator of the loss of self-renewal and pluripotency accompanied by the induction of differentiation in normal and neoplastic hPSCs GFP reporter lines. Automated high content microscopy (HCS) and fluorimetric-based high-throughput plate reader screening (PRS) were used to detect any reduction in pluripotency marker expression. 590 well-established annotated compounds from the NIH Clinical Collection and Canadian Compound Collection were exposed to the cells at 10mM, a level chosen based on previous work, leading to the identification of 11 suitable compounds. Subsequent refinements of the analyses found only thioridazine and mefloquine to possess effective concentration (EC50) values lower than the 10μM target threshold and were defined as candidates for further in-depth evaluation using neoplastic hPSCs and somatic CSCs.
Treatment of normal hPSCs with 10mM thioridazine and mefloquine did not reduce OCT4 levels below those normally observed during BMP4-mediated differentiation, but OCT4 downregulation did occur upon treatment of the neoplastic hPSCs with thioridazine (1mM and 10mM), suggesting that thioridazine may allow the cells to overcome the differentiation block, and therefore could potentially be used as an anti-CSC agent. Indeed, transcriptional analysis found that thioridazine treatment led to an upregulation in 21 of 50 differentiation-associated genes in neoplastic hPSCs. Next, normal (hematopoietic stem and progenitor cells (HSPCs)) and neoplastic (AML (Acute Myeloid Leukaemia)-blast) populations of the human hematopoietic system were assessed, and progenitor assays (colony forming unit (CFU) formation) were undertaken to assess the impact of thioridazine on in vitro clonogenic and multilineage hematopoietic differentiation. Excitingly, thioridazine treatment reduced AML-blast CFU formation while having little effect on HSPC CFU potential and multilineage composition indicating that thioridazine did not alter normal haematopoiesis. An increase in the frequency of granulocytic AML-blast cells was observed with treatment duration suggesting that thioridazine exhibits its specific targeting of AML cells through the induction of differentiation analogous to differentiation-induction demonstrated in neoplastic hPSCs.
Additional screening with a further 2,446 compounds on neoplastic hPSCs screening again identified thioridazine, along with two other phenothiazine compounds; fluphenazine and prochlorperazine, as hits amongst a list of 26 other compounds identified. Alongside these hits where the known anti-leukaemic drugs rapamycin (an mTOR-inhibitor) and lestaurtinib (a tyrosine kinase inhibitor), which reinforces the detection capacity of the screen using the neoplastic hPSC assays. Of the three phenothiazines identified, thioridazine exhibited the lowest EC50 in neoplastic hPSCs making it the best most effective phenothiazine of those tested.
Xenotransplantation studies (Dick, 2008) that functionally define leukaemic stem cells (LSCs) and hematopoietic stem cells (HSCs) were next conducted. HSPCs treated with thioridazine (10μM) displayed the same level of bone marrow (BM) engraftment and splenic engraftment as control vehicle-treated cells, and additionally, multilineage reconstitution capacity was identical between control- and thioridazine-treated hHSCs. Secondary serial transplantation also demonstrated that thioridazine treatment did not affect hHSC self-renewal, but was able to significantly reduced leukaemic disease-initiating AML-LSCs while preserving normal hHSC capacity. In the absence of thioridazine, no difference in the level of leukaemic engraftment of secondary transplant recipients was observed suggesting that continued exposure to thioridazine is necessary to inhibit leukaemogenesis in secondary recipients.
It is known that thioridazine functions through dopamine receptors (DR1-5) (Seeman and Lee) and so DR antagonism was assayed to understand if this was the mechanism through which normal and neoplastic hPSCs are differentially targeted. Initial mRNA analysis of DRs demonstrated their absence in normal hPSCs and the expression of all 5 DRs in neoplastic hPSCs. Additionally, DRs were not found in primitive hHSCs or progenitor populations of cord blood (CB), nor on erythroid, megakaryocytic or lymphoid cells, but were found on monocytes and half the granulocyte population. However, varying levels of all 5 DRs were found in blast cells of 13 AML patients, predominantly in the CD34+/CD14+ compartment, and analysis in triple negative breast cancer cells found DR co-localisation with CD44+CD24−/lo breast CSCs, where previous data has shown that DRs are expressed only at low levels in normal mammary gland tissue but become upregulated in breast cancer (Carlo et al), supporting the use of a drug targeting the dopamine receptors in an additional cancer type. Further research found that DR3- and 5-expressing AML samples contained LSCs that could cause leukaemia in xenotransplants, as compared to AML samples with low levels of DRs, which could not.
Subsequent functional analysis utilised AML cell lines which express DR1-5 at high levels which were then divided into DR+ and DR- fractions. A reduction in blast-CFU generation was observed in the DR+ subfraction treated with thioridazine whereas no reduction was observed in the DR− subfraction treated with thioridazine. Treatment with a DR-D2-family agonist increased the number of AML cells, while treatment with a DR-D1-family agonist reduced AML cell number suggesting that antagonism of D2-family DRs is the main function of thioridazine. Finally, thioridazine’s anti-LSC effect was investigated when combined with conventional AML chemotherapy using cytarabine (AraC), which alone poses significant morbidity and mortality risks at high doses (Estey and Döhner). The combination of thioridazine at 10μM with AraC at 100nM demonstrated the almost complete elimination of AML-blast-CFUs whilst preserving HSPC function, suggesting that at these specified concentrations combined thioridazine/AraC treatment can induce remission and prevent relapse of AML in patients as well as reduce the severe cytotoxic effects associated with high-dose AraC therapy.
The identification of a drug which can preferentially “attack” CSCs is an important finding in current times when growing evidence suggest that CSCs initiate and maintain many tumour types (Chen et al, Driessens et al and Schepers et al) and also exemplifies the role that differentiation-inducing agents can play in anti-CSC therapies. The discovery of targeted chemotherapeutic is also important in terms of the discovery that normal chemotherapeutic agents used to treat prostate cancer can actually imbue normal cells surrounding the tumour site with the capabilities to sustains tumor growth and causes resistance to further treatment. (Sun et al). Further work is now needed to analyse the role of dopamine receptors and their specific targets in other tumour types in order to uncover whether this could be a “magic bullet” for cancer.
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STEM CELLS correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.
Original study from Cell.