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.

Proximity ligation assays for isoform-specific Akt activation in breast cancer identify activated Akt1 as a driver of progression.

The PI3K/Akt signal transduction pathway plays an important role in cancer progression and cell survival. Akt activation is associated with poor outcome in endocrine-treated breast cancer, whereas high levels of cytoplasmic Akt2 are associated with an improved overall survival. Proximity ligation assays (PLAs) were used to determine quantitative expression levels of isoform-specific activation (phosphorylation) of Akt1 and Akt2 in formalin-fixed, paraffin-embedded cell lines and breast cancer tumour tissues in situ. PLAs demonstrated a range of expression in breast cancer samples for total pAkt1 and pAkt2. High levels of pAkt1 were associated with reduced DRFS (HR: 1.45, 95% CI 1.14-1.83, p = 0.002) and OS (HR: 1.42, 95% CI 1.10-1.83, p = 0.007). When PLA results were combined, patients that had high levels of pAkt1 only had a significantly decreased DRFS (HR: 1.92, 95% CI 1.34-2.76, p = 0.005) and OS (HR: 1.94, 95% CI 1.32-2.86, p = 0.008) compared to other patients. Using PLAs to discriminate activation of Akt1 versus Akt2 suggests that Akt1 drives progression in early breast cancers. In cases where both Akt1/Akt2 are activated, Akt2 may act to reverse this effect. Using PLAs, we have measured activation of Akt1 and Akt2 proteins separately in situ in FFPE breast cancer samples.

In situ detection of HER2:HER2 and HER2:HER3 protein-protein interactions demonstrates prognostic significance in early breast cancer.

HER2 overexpression/amplification is linked with poor prognosis in early breast cancer. Co-expression of HER2 and HER3 is associated with endocrine and chemotherapy resistance, driven not simply by expression but by signalling via HER2:HER3 or HER2:HER2 dimers. Proximity ligation assays (PLAs) detect protein-protein complexes at a single-molecule level and allow study of signalling pathways in situ. A cohort of 100 tumours was analyzed by PLA, IHC and FISH. HER complexes were analyzed by PLA in a further 321 tumours from the BR9601 trial comparing cyclophosphamide, methotrexate and fluorouracil (CMF) with epirubicin followed by CMF (epi-CMF). The relationships between HER dimer expression and RFS and OS were investigated, and multivariate regression analysis identified factors influencing patient prognosis. PLA successfully and reproducibly detected HER2:HER2 and HER2:HER3 protein complexes in vivo. A significant association (P < 0.00001) was identified between HER2 homodimerization and HER2 gene amplification. Following a minimum p value approach high levels of HER2:HER2 dimers were significantly associated with reduced relapse-free (RFS; hazard ratio = 1.72, 95% confidence interval 1.15-2.56, P = 0.008) and overall survival (OS HR = 1.69 95% CI = 1.09-2.62, P = 0.019). Similarly, high levels of HER2:HER3 dimers were associated with reduced RFS (HR = 2.18, 95% CI = 1.46-3.26, P = 0.00016) and OS (HR = 2.21, 95% CI = 1.41-3.47, P = 0.001). This study demonstrates that in situ detection of HER2 and HER2:3 protein:protein complexes can be performed robustly and reproducibly in clinical specimens, provides novel prognostic information and opens a significant novel opportunity to probe the clinical impact of cellular signalling processes.