Downregulation of histone H2A and H2B pathways is associated with anthracycline sensitivity in breast cancer.

BACKGROUND: Drug resistance in breast cancer is the major obstacle to effective treatment with chemotherapy. While upregulation of multidrug resistance genes is an important component of drug resistance mechanisms in vitro, their clinical relevance remains to be determined. Therefore, identifying pathways that could be targeted in the clinic to eliminate anthracycline-resistant breast cancer remains a major challenge. METHODS: We generated paired native and epirubicin-resistant MDA-MB-231, MCF7, SKBR3 and ZR-75-1 epirubicin-resistant breast cancer cell lines to identify pathways contributing to anthracycline resistance. Native cell lines were exposed to increasing concentrations of epirubicin until resistant cells were generated. To identify mechanisms driving epirubicin resistance, we used a complementary approach including gene expression analyses to identify molecular pathways involved in resistance, and small-molecule inhibitors to reverse resistance. In addition, we tested its clinical relevance in a BR9601 adjuvant clinical trial. RESULTS: Characterisation of epirubicin-resistant cells revealed that they were cross-resistant to doxorubicin and SN-38 and had alterations in apoptosis and cell-cycle profiles. Gene expression analysis identified deregulation of histone H2A and H2B genes in all four cell lines. Histone deacetylase small-molecule inhibitors reversed resistance and were cytotoxic for epirubicin-resistant cell lines, confirming that histone pathways are associated with epirubicin resistance. Gene expression of a novel 18-gene histone pathway module analysis of the BR9601 adjuvant clinical trial revealed that patients with low expression of the 18-gene histone module benefited from anthracycline treatment more than those with high expression (hazard ratio 0.35, 95 % confidence interval 0.13-0.96, p = 0.042). CONCLUSIONS: This study revealed a key pathway that contributes to anthracycline resistance and established model systems for investigating drug resistance in all four major breast cancer subtypes. As the histone modification can be targeted with small-molecule inhibitors, it represents a possible means of reversing clinical anthracycline resistance. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00003012 . Registered on 1 November 1999.

Molecular characterisation of isogenic taxane resistant cell lines identify novel drivers of drug resistance.

BACKGROUND: Taxanes such as paclitaxel and docetaxel are used successfully to treat breast cancer, usually in combination with other agents. They interfere with microtubules causing cell cycle arrest; however, the mechanisms underlying the clinical effects of taxanes are yet to be fully elucidated. METHODS: Isogenic paclitaxel resistant (PACR) MDA‒MB‒231, paclitaxel resistant ZR75‒1 and docetaxel resistant (DOCR) ZR75‒1 cell lines were generated by incrementally increasing taxane dose in native cell lines in vitro. We used aCGH analysis to identify mechanisms driving taxane resistance. RESULTS: Taxane resistant cell lines exhibited an 18-170 fold increased resistance to taxanes, with the ZR75-1 resistant cell lines also demonstrating cross resistance to anthracyclines. Paclitaxel treatment of native cells resulted in a G2/M block and a decrease in the G1 phase of the cell cycle. However, in the resistant cell lines, minimal changes were present. Functional network analysis revealed that the mitotic prometaphase was lost in the resistant cell lines. CONCLUSION: This study established a model system for examining taxane resistance and demonstrated that both MDR and mitosis represent common mechanism of taxane resistance.

Expression of activated type I receptor tyrosine kinases in early breast cancer.

Overexpression of EGFR, HER2 and HER3 are known to be associated with poor outcome in breast cancer. Few studies have examined the clinical impact of activation of these proteins. In the present study, we evaluated EGFR, HER2 and HER3 and the activated (phosphorylated) forms of these proteins in patients with early breast cancer. EGFR, HER2, HER3, pEGFR, pHER2 and pHER3 expression was determined by immunohistochemical analysis of tissue microarrays constructed from tumours within the Edinburgh Breast Conservation Series (BCS). The BCS represents a fully-documented consecutive cohort of 1,812 patients treated by breast conservation surgery in a single institution. Our results demonstrate overexpression of HER2 and pHER2 to be associated with a significant reduction in overall survival (OS) (HR: 1.66, 95 % CI 1.22-2.26, p = 0.001 and HR: 1.57, 95 % CI 1.22-2.03, p = 0.001, respectively) and distant relapse-free survival (DRFS) (HR: 1.63, 95 % CI 1.23-2.18, p = 0.001 and HR: 1.55, 95 % CI 1.23-1.97, p = 0.0002, respectively). Paradoxically, expression of pEGFR was associated with a significantly improved OS (HR: 0.67 95 % CI 0.50-0.91, p = 0.01) and DRFS (HR: 0.73, 95 % CI 0.56-0.96, p = 0.025). Expression of activated EGFR/HER2 provides additional information on ER positive breast cancer patients and suggests alternative treatment for those in this subgroup.

Deregulation of the spindle assembly checkpoint is associated with paclitaxel resistance in ovarian cancer.

BACKGROUND: Ovarian cancer is the leading gynecologic cancer diagnosed in North America and because related symptoms are not disease specific, this often leads to late detection, an advanced disease state, and the need for chemotherapy. Ovarian cancer is frequently sensitive to chemotherapy at diagnosis but rapid development of drug resistance leads to disease progression and ultimately death in the majority of patients. RESULTS: We have generated paclitaxel resistant ovarian cell lines from their corresponding native cell lines to determine driver mechanisms of drug resistance using gene expression arrays. These paclitaxel resistant ovarian cells demonstrate: (1) Increased IC50 for paclitaxel and docetaxel (10 to 75-fold) and cross-resistance to anthracyclines (2) Reduced cell apoptosis in the presence of paclitaxel (3) Gene depletion involving mitotic regulators BUB1 mitotic checkpoint serine/threonine kinase, cyclin BI (CCNB1), centromere protein E (CENPE), and centromere protein F (CENPF), and (4) Functional data validating gene depletion among mitotic regulators. CONCLUSIONS: We have generated model systems to explore drug resistance in ovarian cancer, which have revealed a key pathway related to the spindle assembly checkpoint underlying paclitaxel resistance in ovarian cell lines.

A four gene signature of chromosome instability (CIN4) predicts for benefit from taxanes in the NCIC-CTG MA21 clinical trial.

Recent evidence demonstrated CIN4 as a predictive marker of anthracycline benefit in early breast cancer. An analysis of the NCIC CTG MA.21 clinical trial was performed to test the role of existing CIN gene expression signatures as prognostic and predictive markers in the context of taxane based chemotherapy.RNA was extracted from patients in cyclophosphamide, epirubicin and flurouracil (CEF) and epirubicin, cyclophosphamide and paclitaxel (EC/T) arms of the NCIC CTG MA.21 trial and analysed using NanoString technology.After multivariate analysis both high CIN25 and CIN70 score was significantly associated with an increased in RFS (HR 1.76, 95%CI 1.07-2.86, p=0.0018 and HR 1.59, 95%CI 1.12-2.25, p=0.0096 respectively). Patients whose tumours had low CIN4 gene expression scores were associated with an increase in RFS (HR: 0.64, 95% CI 0.39-1.03, p=0.06) when treated with EC/T compared to patients treated with CEF.In conclusion we have demonstrated CIN25 and CIN70 as prognostic markers in breast cancer and that CIN4 is a potential predictive maker of benefit from taxane treatment.

A four gene signature predicts benefit from anthracyclines: evidence from the BR9601 and MA.5 clinical trials.

Chromosome instability (CIN) in solid tumours results in multiple numerical and structural chromosomal aberrations and is associated with poor prognosis in multiple tumour types. Recent evidence demonstrated CEP17 duplication, a CIN marker, is a predictive marker of anthracycline benefit. An analysis of the BR9601 and MA.5 clinical trials was performed to test the role of existing CIN gene expression signatures as predictive markers of anthracycline sensitivity in breast cancer. Univariate analysis demonstrated, high CIN25 expression score was associated with improved distant relapse free survival (DRFS) (HR: 0.74, 95% CI 0.54-0.99, p = 0.046). High tumour CIN70 and CIN25 scores were associated with aggressive clinicopathological phenotype and increased sensitivity to anthracycline therapy compared to low CIN scores. However, in a prospectively planned multivariate analysis only pathological grade, nodal status and tumour size were significant predictors of outcome for CIN25/CIN70. A limited gene signature was generated, patients with low tumour CIN4 scores benefited from anthracycline treatment significantly more than those with high CIN4 scores (HR 0.37, 95% CI 0.20-0.56, p = 0.001). In multivariate analyses the treatment by marker interaction for CIN4/anthracyclines demonstrated hazard ratio of 0.35 (95% CI 0.15-0.80, p = 0.012) for DRFS. This data shows CIN4 is independent predictor of anthracycline benefit for DRFS in breast cancer.