Jackie Perry

The Association of CCND1 Overexpression and Cisplatin Resistance in Testicular Germ Cell Tumors and Other Cancers

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Phase I and Pharmacokinetic Study of Intravenous Irinotecan Plus Oral Ciclosporin in Patients With Fluorouracil-Refractory Metastatic Colon Cancer

Purpose: To assess the safety and toxicity profile of escalating doses of intravenous irinotecan, in combination with a fixed dose of oral ciclosporin (Cs) and to determine the pharmacokinetic profile of irinotecan and its metabolites. Patients and Methods: Patients with fluorouracil-refractory metastatic colorectal cancer received escalating doses of intravenous irinotecan from 40 to 125 mg/m2 every 2 weeks in combination with a fixed dose of oral Cs (5 mg/kg bid for 3 days). Pharmacokinetic analysis of plasma irinotecan and its metabolites SN38 and SN38G was performed during paired cycles with and without Cs. Results: Thirty-seven patients were treated. Dose-limiting toxicity of grade 4 neutropenia was seen at an irinotecan dose of 125 mg/m2. There was no grade 4 diarrhea, and only one patient experienced grade 3 diarrhea. Toxicities caused by Cs were generally mild. Pharmacokinetic studies demonstrated that irinotecan clearance was reduced from 13.4 to 5.8 L/h/m2 and area under the curve (AUC)0-tn was ...

A Synergistic Interaction between Lapatinib and Chemotherapy Agents in a Panel of Cell Lines Is Due to the Inhibition of the Efflux Pump BCRP

Lapatinib is a specific HER1 and 2 targeted tyrosine kinase inhibitor now widely used in combination with chemotherapy in the clinical setting. In this work, we investigated the interactions between lapatinib and specific chemotherapy agents (cisplatin, SN-38, topotecan) in a panel of cell lines [breast (n = 2), lung (n = 2), testis (n = 4)]. A high-sensitivity cell proliferation/cytotoxicity ATP assay and flow cytometry were used to determine cell viability, apoptosis, and the effect of the drugs on cell-cycle distribution. CalcuSyn analysis was employed to formally identify synergistic interactions between drugs. Intracellular concentrations of SN-38 were measured using a novel high-performance liquid chromatography (HPLC) technique. Flow cytometry and HPLC techniques were used to identify the effect of lapatinib on drug influx and efflux pumps, using specific substrates and inhibitors of these pumps. Results showed significant synergy between SN-38, and lapatinib in the majority of cell lines (combination index < 0.75), associated with increased apoptosis. This synergy was not universal but, when observed (Susa S/R, H1975, H358, and MDA-MB-231 cell lines), was related to SN-38 intracellular accumulation (2.2- to 4.8-fold increase, P < 0.05 for each), attributable to the inhibition of the breast cancer–related protein (BCRP) efflux pump by lapatinib. Flow cytometry analysis showed that lapatinib (10 μmol/L) inhibited the efflux of mitoxantrone, a specific substrate of the BCRP pump, in a manner similar to fumitremorgin C, a known BCRP inhibitor, confirming lapatinib as a BCRP inhibitor. This work shows that lapatinib has a direct inhibitory effect on BCRP accounting for the synergistic findings. The synergy is cell line dependent and related to the activity of specific efflux pumps. Mol Cancer Ther; 9(12); 3322–9. ©2010 AACR.

Identification of genomic changes associated with cisplatin resistance in testicular germ cell tumor cell lines

Since the introduction of cisplatin into the clinic, the treatment of patients with a variety of solid tumors including testicular germ cell tumors, ovarian and lung cancers, has dramatically improved. One of the main causes for therapeutic failure in these malignancies is the development of drug resistance. Testicular germ cell tumors (TGCTs), the most common malignancy in young men, exhibit extreme sensitivity to cisplatin‐based chemotherapy, making them an ideal model for investigating the mechanisms of cisplatin chemo‐sensitivity and resistance. TGCT development and pathogenesis have been well studied but little is known about the genetic background in chemo‐resistant cases. We investigated genomic differences between three TGCT parental cell lines and their cisplatin resistant derivatives. Using 10K single nucleotide polymorphism (SNP) microarray analysis, we identified two small chromosomal regions with consistent copy number changes across all three pairs of resistant cell lines. These were an 8.7 Mb region at 6q26‐27, which displayed consistent copy number gain and a 0.3 Mb deletion involving 4 SNPs at 10p14. Both the chromosomal gain and loss were confirmed by fluorescence in situ hybridization. The significance of these regions should be further investigated as they may contain key genes involved in the development of chemo‐ resistance to cisplatin‐based treatment in TGCTs and other cancers.

Development and validation of an ultra-high performance LC-MS/MS assay for intracellular SN-38 in human solid tumour cell lines: comparison with a validated HPLC-fluorescence method.

A simple and rapid ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS) method has been developed for measuring intracellular concentrations of the anticancer agent 7-ethyl-10-hydroxycamptothecin (SN-38) in tumour cells using camptothecin (CPT) as internal standard. SN-38 extraction was carried out using acidified acetonitrile. SN-38 and CPT were separated on a PFP column using gradient elution with acidified water and acetonitrile. SN-38 and CPT were quantified using a triple quadrupole mass spectrometry system. Least square regression calibration lines were obtained with average correlation coefficients of R(2)=0.9993±0.0016. The lower limit of detection (LOD) and lower limit of quantification (LOQ) for SN-38 were 0.1 and 0.3ng/ml, respectively. CPT recovery was 98.5±13% and SN-38 recoveries at low quality control (LQC, 5ng/ml) and high quality control (HQC, 500ng/ml) were 89±6% and 95±8%, respectively. The intra- and inter-day imprecision for LQC was 5.8 and 8.5%, and for HQC was 6.3 and 4.4%, respectively. The method was compared to a validated high performance liquid chromatography-fluorescent method. In addition, the method has been successfully applied to determine the intracellular accumulation of SN-38 investigating the transport through ABCB1 (P-gp) and ABCG2 (BCRP) efflux pumps in colorectal cancer cell lines.

The histone deacetylase inhibitor UCL67022 has potent activity in multiple myeloma and non-Hodgkin lymphoma pre-clinical models.

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Abstract 1792: The potential role of targeting HER1-4 in bladder cancer

Introduction: The survival of patients with invasive bladder cancer is short, suggesting an urgent need for new treatments. Given that HER family receptor tyrosine kinases (TK) are associated with tumor stage/grade and outcome in this malignancy (Chow et al, Clin Cancer Res, 2001), approaches using small molecule inhibitors of HER receptors may be of value. In the present study we have investigated the potential utility and mechanism of action of Gefitinib (HER1 inhibitor), CP-724714(HER2 inhibitor), Lapatinib(dual HER1/2 inhibitor) and Canertinib (pan-HER inhibitor) in a panel of human bladder cancer cell lines. Methods: Four bladder cancer cell lines were used (T24, J82, H1376, 647V). HER 1-4 receptor protein and transcript were determined by Western blot and RT-PCR respectively. An ATP assay was used to determine the cytotoxic effect of HER targeted TKIs, with effects on cell cycle distribution and apoptosis determined by flow cytometric methods. A cell line with high receptor expression was selected for RNAi-silencing experiments targeting HER2 using lentivectors to determine the effects of specific HER2 silencing compared to the effects of HER2 inhibition. The differential effects of HER2 inhibition or silencing on downstream signalling was studied using western blot analysis and LC-MS/MS based methods for Akt activity and phosphoprotein profiling. Results: The expression levels of HER1-4 members varied between the cell lines. Sensitivity to TKIs also varied and was not correlated with HER1-4 expression. However, the dual (Lapatinib) and pan (Canertinib) -HER inhibitors were more potent (up to 30-fold) than the single HER inhibitors Gefitinib and CP-724714. Cell cycle analysis typically showed an increase in the G2/M population with all treatments, with a clear increase in apoptotic cells (up to 70%) observed only with Lapatinib. RNAi mediated HER2-knockdown in T24 cells was confirmed by western blotting and RT-PCR. With the exception of Gefitinib, sensitivity to TKIs was not altered by HER2 knockdown, with EC50 values for the HER2 TKI CP-724714 in parental and HER2-knockdown cells of 36.4 ± 14.5 vs 31.5 ± 10.3 µM respectively. Both HER2 RNA-silencing and inhibition by TKIs resulted in decreased downstream signalling (40% and 70% respectively), based on PI3K activity and phosphorylation of AKT. Phosphoproteomic analysis of TKI inhibited or HER2-knockdown cells showed common phosphoproteins (AHNAK, ASF-1) that were altered with all treatments, and others that were specific for HER2 silencing, HER2 TKI or HER2 mAb (Trastuzumab) exposure. Similar studies are now being performed in a second cell line. Conclusions: TKIs targeting HER1-4 receptors had a clear effect on the viability of bladder cancer cells. Both HER2-RNA silencing and TKIs resulted in decreased PI3K pathway activity. Phosphoproteins altered by these treatments highlight specific kinases that might represent potential therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1792. doi:1538-7445.AM2012-1792

Abstract 3256: Sphingolipid regulation by sphingosine kinase anchoring protein (SKAP) and its implication in cancer

Background: Sphingolipids are important in cancer cell signalling. Sphingosine 1 phosphate (S1P) promotes cell survival and resistance to apoptosis, while S1P precursors ceramide (CER) and sphingosine (SPH), mediate antiproliferative and apoptotic responses. S1P is generated from SPH by sphingosine kinase (SK) enzymes (SK1 and SK2), with SK activity and localisation regulated by other proteins, including PKC, PKA and a SK anchoring protein (SKAP) that has been reported to negatively regulate SK1 activity in fibroblasts. S1P localisation is thought to play an important role in its function. Based on our preliminary observation in primary AML cells that SKAP expression resulted in an increase in S1P, we have investigated the effect of SKAP transfection on S1P production and localisation. Methods: K562, (and for some confirmatory experiments MCF-7), cells were transfected with the SKAP gene using standard techniques. SKAP is normally silenced in both cell lines. Transfection was confirmed by RNA expression. Intracellular and extracellular S1P and SPH, and intracellular SK activity (based on the production of C17 S1P from C17 SPH, an unnatural SPH that is a SK substrate) in intact cells were measured by LC-MS/MS. Phorbol 12-myristate 13-acetate (PMA) was used to induce membrane associated SK function, and MK-571 and fumitremorgen C (FTC) were used to block S1P efflux through ABCC1 and ABCG2 efflux pumps, respectively. Chemosensitivity to doxorubicin and imatinib in transfected cells was also studied. Results: K562 cells transfected with the SKAP gene showed a 2.5 fold increase in intracellular and extracellular levels of basal S1P compared to vector alone control. (In MCF-7 cells SKAP transfection resulted in an almost 10-fold increase in S1P). Further studies in K562 cells confirmed a significant increase in intracellular SK activity in SKAP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3256. doi:1538-7445.AM2012-3256