We previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered. In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours. Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

Background: Renal cell carcinoma patients respond poorly to conventional chemotherapy, this unresponsiveness may be attributable to multidrug resistance (MDR). The mechanisms of MDR in renal cancer are not fully understood and the specific contribution of ABC transporter proteins which have been implicated in the chemoresistance of various cancers has not been fully defined in this disease.Methods: In this retrospective study the expression of two of these transporter efflux pumps, namely MDR-1 P-gp (ABCB1) and MRP-1 (ABCC1) were studied by immunohistochemistry in archival material from 95 renal cell carcinoma patients.Results: In the first study investigating MDR-1 P-gpandMRP-1 protein expression patterns in renal cell carcinoma patients, high levels of expression of both efflux pumps are observed with 100% of tumours studied showing MDR-1 P-gpandMRP-1 positivity.Conclusion: Although these findings do not prove a causal role, the high frequency of tumours expressing these efflux pumps suggests that they may be important contributors to the chemoresistance of this tumour type.

Markers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion) and Clone #8 (low invasion) using proteomic profiling of an in vitro model of pancreatic cancer. Using 2D-DIGE followed by MALDI-TOF MS, two clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with high and low invasive capacities were incubated on matrigel 24 hours prior to analysis to stimulate cell-ECM contact and mimic in vivo interaction with the basement membrane. Sixty proteins were identified as being differentially expressed (> 1.2 fold change and p ≤ 0.05) between Clone #3 and Clone #8. Proteins found to have higher abundance levels in the highly invasive Clone #3 compared to the low invasive Clone #8 include members of the chaperone activity proteins and cytoskeleton constituents whereas metabolism-associated and catalytic proteins had lower abundance levels. Differential protein expression levels of ALDH1A1, VIM, STIP1 and KRT18 and GAPDH were confirmed by immunoblot. Using RNAi technology, STIP1 knockdown significantly reduced invasion and proliferation of the highly invasive Clone #3. Knockdown of another target, VIM by siRNA in Clone #3 cells also resulted in decreased invasion abilities of Clone #3. Elevated expression of STIP1 was observed in pancreatic tumour tissue compared to normal pancreas, whereas ALDH1A1 stained at lower levels in pancreatic tumours, as detected by immunohistochemistry. Identification of targets which play a role in the highly invasive phenotype of pancreatic cancer may help to understand the biological behaviour, the rapid progression of this cancer and may be of importance in the development of new therapeutic strategies for pancreatic cancer.

With a five-year survival rate of 9%, pancreatic ductal adenocarcinoma (PDAC) is the deadliest of all cancers. The rapid mortality makes PDAC difficult to research, and inspires a resolve to create reliable, tractable cellular models for preclinical cancer research. Organoids are increasingly used to model PDAC as they maintain the differentiation status, molecular and genomic signatures of the original tumour. In this paper, we present novel methodologies and experimental approaches to develop PDAC organoids from PDX tumours, and the simultaneous development of matched primary cell lines. Moreover, we also present a method of recapitulating primary cell line cultures to organoids (CLOs). We highlight the usefulness of CLOs as PDAC organoid models, as they maintain similar transcriptomic signatures as their matched patient-derived organoids and patient derived xenografts (PDX)s. These models provide a manageable, expandable in vitro resource for downstream applications such as high throughput screening, functional genomics, and tumour microenvironment studies.

Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.