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A RSKy Target Pays off for Breast Cancer



Original article from STEM CELLS

Triple-negative breast cancer (TNBC) refers to any breast cancer that does not express the genes for estrogen receptor (ER), progesterone receptor (PR) or Her2/neu andtherefore does not respond to conventional therapeutic approaches.   These tumours become resistant to chemotherapeutic agents and are prone to recurrence, which has been linked to the presence of high numbers of CD44+/CD24 tumour initiating cells (TICs) which are intrinsically resistant to traditional chemotherapy and radiotherapy (Creighton et al, Li et al and Philips et al).  A further concern is that the percentage of TICs increases following chemotherapeutic treatment of breast cancers with agents such as paclitaxel (Creighton et al and Fillmore and Kuperwasser).  This suggests that the targeting of CD44+ TICs may be of value to the treatment of TNBC.   A previous study has shown that the transcription factor YB-1 can regulate the TIC phenotype in TNBC, increasing TIC marker (CD44 and CD49f (or ITGA6)) expression, mammosphere formation and drug resistance (To et al).  YB-1 is phosphorylated and activated, leading to nuclear translocation and transcriptional activation, by the p90 ribosomal S6 kinases (RSK) (To et aland Stratford et al) suggesting that these kinases may be a viable therapeutic target for TNBC.   Now, researchers from the laboratory of Sandra E. Dunn at the University of British Columbia, Vancouver, Canada have reported that blocking the activation of YB-1 via RSK inhibition could be an alternative approach to combating relapse by eliminating TICs present in TNBC (Stratford and Reipas et al).

Initial studies utilised a stable YB-1 overexpressing MDA-MB-231 breast cancer cell line and following injection in the mammary fat pad of NOD/SCID mice YB-1 overexpressing cells were noted to have an increased growth rate in comparison to control cells, leading to tumours of much greater volume.   YB-1 cells isolated from these tumours had increased CD49f and CD44 expression and phosphorylation of YB-1 itself was increased, suggesting higher levels of TICs.   However, addition of a cell permeable, ATP-competitive inhibitor of the four isoforms of RSK (BI-D1870) led to a reduction in YB-1 phosphorylation and inhibited YB-1+ cell growth.   In vitro kinase assays found that RSK1 and 2 could phosphorylate YB-1 at Ser102 and that BI-D1870 could inhibit this.   Overall this initial data suggested that YB-1 drives tumour formation leading to tumours with higher levels of TICs which are sensitive to RSK-inhibition.

RSK and the affect of its inhibition were then assessed in the context of TNBC using the SUM149 cell line which was generated from a primary human breast cancer.   Expression of RSK siRNA led to a 90% reduction in RSK and phospho-YB-1 at 72 hours, with 10 days of siRNA expression allowing for near complete growth inhibition.   RSK2 inhibition was noted to lead to a stronger effect than RSK1, and an unbiased screen of siRNAs targeting >700 kinases in 20 breast cancer cell line models representing the major subtypes of the disease (Brough et al) confirmed that RSK2 inhibition selectively suppressed the TNBC models and was one of the very limited number of genes that was able to elicit such a TNBC-specific effect.    Consequently, growth suppression of TNBC cell lines in response to RSK inhibitors was assessed, and it was found that BI-D1870 treatment for 10 days led to over 90% reduction in growth rate of SUM149 cells, concomitant with a reduction in phospho-YB1.   Specificity of this effect to YB-1 was observed by the partial rescue of this growth inhibition by the over-expression of phospho-YB-1 in inhibitor treated cells.   RSK inhibition also induced apoptosis as demonstrated by PI uptake, phospho-H2AX, PARP cleavage and Annexin-V staining, while any resulting cells following BI-D1870 treatment were not deemed to be resistant to the drug as these cells failed to grow in clonogenic assays.

It is understood that TICs can be induced by YB-1 binding to the CD44 promoter in a phosphorylation-dependent manner leading to increased CD44 expression (To et aland Stratford et al).   Unfortunately, many chemotherapeutic agents, such as paclitaxel (a mitotic inhibitor), mediate an increase in CD44 expression (Creighton et al)which is thought to play a key role in drug resistance and recurrence in TNBC.   Interestingly, it was discovered that treatment with RSK antagonist BI-D1870 inhibited the nuclear translocation of phospho-YB-1 and CD44 promoter activity in SUM149 cells leading to decreased CD44 mRNA and a reduction in CD44 expressing cells and, importantly, combined paclitaxel and BI-D1870 treatment reduced the paclitaxel –mediated induction of CD44.

In the course of the investigations it was also uncovered that CD44+ cells were more proliferative than CD44- cells as shown through higher levels of mitosis, based on Hoechst staining and phospho-histone H3 detection.   This higher proliferative rate was subdued by BI-D1870, suggesting that RSK inhibition could repress TICs ability to replicate.   CD44+ cells also form mammospheres to a greater capacity than CD44- cells and, correlative to the decrease in CD44+, BI-D1870 suppressed mammosphere formation by 80-100% in MDA-MB-231 and SUM149 cells also leading to the regression of established mammospheres.   CD44+CD24- TICS isolated by fluorescence-activated cell sorting (FACS) were found to also have high levels of phospho-RSK and phospho-YB-1, and treatment of these cells with BI-D1870 reduced their growth rate by over 90% within 72 hours and induced apoptosis.   The effect of RSK inhibition in normal cells was also assessed and no suppressive effects on growth or differentiation were found in HSCs or normal breast epithelial cells.

The role of RSK inhibition on tumour formation was assessed through a transient RSK2 knockdown in homogenous MDA-MB-231 CD44HiCD24Lo TICs.   At 48 hours, RSK2 was decreased over 80% and this led to the loss of CD44 protein expression.   Injection of these cells into NOD/SCID mice led to a 40% tumour development rate at 24 days post-injection, as compared to a 100% tumour development rate in mice injected with control MDA-MB-231 cells.   At 3 weeks, upon loss of RSK2 inhibition in the injected TICs, all mice eventually developed tumours which grew at equivalent rates to the control but gave rise to tumours approximately half the size, overall suggesting that RSK inhibition blocks TNBC cell growth partly through loss of TICs.

Further investigation of patient samples found that breast cancer patients with high RSK2 expression had significantly worse survival outcomes and RSK2 levels were highest in tumors of the basal-like subtype and in those of the highest grade.   This is an important point as the majority of basal-like breast cancers are also triple negative in terms of expression of cell surface receptor proteins (Foulkes et al).   Further analysis of 18 high-grade breast cancers found that phospho-RSK significantly correlated with phospho-YB-1, and in more than half the cases CD44 expression correlated with phospho–RSK/phospho-YB-1.   Analysis of normal breast tissues failed to find this overlap suggesting that invivo the RSK/YB-1/CD44 pathway is activated in TNBC.

Moving from cell lines to patient samples the authors have provided strong data suggesting that YB-1 can drive the initiation of tumours which contain high levels of CD44+ TICS, that TNBCs are dependent on RSK signalling to sustain growth and that RSK inhibitors can block the growth of TNBC cells through the loss of TICs.   Importantly, inhibition of RSK does not appear to affect normal tissues and reduces thepaclitaxel-mediated upregulation of CD44.   Overall this exciting study suggests a likely therapeutic target for the treatment of TNBC.



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STEM CELLS correspondent Stuart Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.