RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation

Abstract

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.

Introduction

Pancreatic adenocarcinoma is one of the most lethal and poorly understood human malignancies. The 5-year survival rate is between 1% and 4% [1], mainly due to the lack of effective therapies. Conventional treatment used to treat pancreatic cancer such as surgery, chemotherapy, radiation or combinations of these have had very little effect on the overall survival of this disease [2].

Hop (Hsp70/Hsp90-organising protein) or STIP1 (stress-induced phosphoprotein 1) mediates the association, and forms a physical complex with the molecular chaperones heat shock proteins (Hsp) Hsp70 and Hsp90 [3], [4] which is dependent on the hydrolysis of ATP and ADP/ATP exchange. Hop optimises the functional co-operation between Hsp70 and Hsp90 which requires highly co-ordinated interactions for the folding and conformational regulation of a variety of proteins [5]. The molecular chaperone complex of Hop, Hsp70 and Hsp90 is involved in the folding and maturation of key regulatory proteins involved in cell viability [6], such as steroid hormone receptors, transcription factors and kinases (some of which are involved in cancer progression). The role of Hop in tumour progression is not fully elucidated; however, Hop has been shown to be over expressed in colonic carcinoma cells [7] and in hepatocellular carcinoma (HCC) [8]. Hop is secreted by and shown to induce proliferation in glioma tumour cells through MAPK and P13K pathways [9]. Hop has also been implicated in the controlled death process. Bredemeyer et al. [10] confirmed Hop as a substrate of the apoptotic protease granzyme B (GzmB), as Hop was directly cleaved by GzmB in vitro and in cells undergoing GzmB-induced apoptosis. Expression of the two cleavage fragments of Hop did not induce cell death; however, cleavage of Hop by GzmB did result in loss of Hop function in vitro.

There is increasing evidence that heat shock proteins play an important role in the development, maintenance and progression of cancer [11]. Heat shock proteins such as, Hsp90 and Hsp70 have previously been implicated in pancreatic cancer [12], [13]. Hsps are highly conserved molecules, which protect cells from various environmental damages, including stress and carcinogenesis [14] and are activated by the ubiquitin–proteasome pathway. The covalent attachment of ubiquitin to protein regulates a number of cellular processes including responses to stress. This process is responsible for selective targeting of proteins for degradation. The induced Hsps play roles as cellular chaperones, and many modulate apoptotic pathways, particularly those involving mitochondria, conferring protection from stressful stimuli including chemotherapeutic agents [15]. Hsp90 client proteins include mutated p53, Bcr-Abl, Raf-1, Akt, HER2 (ErbB2) and hypoxia inducible factor-1alpha (HIF-1alpha) [16].

We previously profiled Hop expression in an in vitro pancreatic cancer cell line invasion model and pancreatic tumour tissue specimens [17]. In the present study, we hypothesised that modulation of Hop expression could alter invasion and affect the activity of Hsp90 and its client proteins; suppression of Hop by siRNA reduced invasion through decreased expression of MMP-2. We further show that modulation of Hop alters the activities of representative Hsp90 client proteins HER2, Bcr-Abl, c-MET and v-Src. This study reports that Hop is likely to play a role in invasion of pancreatic cancer cells, and possibly contribute to the highly aggressive metastatic phenotype of pancreatic cancer. In addition our findings support Hop as a potential therapeutic target for the treatment of pancreatic cancer.