Investigation of the chemoresistance mechanisms in subpopulations of renal cell carcinoma cells

Abstract

'Investigation of the Chemoresistant Mechanisms inSubpopulations of Renal Cell Carcinoma Cells'Background/ Introduction: Kidney cancer accounts for approximately 2-3% of all cancers worldwide. Renal cell carcinoma (RCC) is the commonest malignant renal tumour with 7,500 cases diagnosed annually resulting in over 3,700 deaths in the UK. The only curative treatment is surgery for the 50% presenting with early-stage disease. Patients with metastatic disease have a 5 year survival rate less than 10%, due to chemo-radiotherapy resistance. Newer drugs, so-called 'targeted' agents such as temsirolimus, sunitinib and sorafenib, have shown some activity against metastatic RCC. However, this is not longstanding and tumours inevitably progress. A key mechanism in chemoresistance of RCC is the ability of RCC cells to efflux chemotherapeutic agents via multidrug resistant pumps (MDR), where MDR expression has been correlated with tumour progression and poor prognosis. It has recently been shown that RCC specimens contain a side population (SP), as defined by the constituent cell's ability to efflux Hoechst 33342 dye and cytotoxic agents. The SP is enriched for cancer stem-like cells. The hypothesis tested herein is that the cells resident within the SP are the root cause of chemoresistance, and that characterisation of these cells will facilitate the understanding of the biology of this process and in addition, help to develop novel therapeutic approaches. Moreover, recent reports have suggested that the 'targeted' agents used in metastatic RCC may also affect MDR function, which forms the basis of these studies. Methods: Eight primary and metastatic human RCC lines (2220R, 2245R, 2246R, 2247R, ACHN, A498, Caki-1 and Caki-2) were assessed for the presence of Hoechst 33342 SP sub-populations and their growth characteristics were determined in vitro. Candidate renal sub-populations were sub-fractionated using Hoechst 33342 incubation and FACS- based separation, and treated with the chemotherapeutic agents docetaxel, paclitaxel, adriamycin, cisplatin, 5-fluorouracil and etoposide. IC50 values were determined by SRB assay and drug efflux by FACS and fluorescence microscopy. The novel targeted agents (temsirolimus, sunitinib and sorafenib) in RCC were interrogated as potential MDR modulator agents using FACS and SRB assays in combination with selected chemotherapy agents. Western blot experiments were performed to define the dosing strategy. qRT-PCR experiments investigated MDR expression levels in the SP compared to the NSP. Key signalling pathways involved in the conventional mechanism of action of the various targeted agents defined the doses where activity was demonstrated i.e. mTOR inhibition. Results: Characteristic verapamil sensitive SPs were seen in 5 of the 8 human RCC cell lines. The 2245R cell line had the highest SP (6.0±4.6%), and was chosen for further studies. The IC50 values for the SP, compared to the NSP, were significantly higher with docetaxel, paclitaxel, etoposide and adriamycin (5.4 vs. 3.3nM, 41.8 vs. 13.4nM, 11.6 vs. 8.8µM and 89.6 vs. 47.3nM respectively). There was no difference seen in these subpopulations treated with cisplatin or 5-fluorouracil (which are not MDR pump substrates). Retention of adriamycin fluorescence following treatment was significantly lower in the SP when measured both quantitively using FACS, and assessed qualitatively using microscopy (p

Date of Award	1 Aug 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Brown (Supervisor) & Noel Clarke (Supervisor)