Recent Progress 2015

In addition to the cancer cells, solid tumours also contain a range of “normal” cells, such as immune cells, endothelial cells and fibroblasts, which together form the tumour stroma. There has been a general appreciation that understanding the role of these infiltrating cells on tumour progression and response to therapy is needed in order to develop more efficient treatments. In the Systems Oncology Group we aim to determine how tumour cells signal with cells in the stroma and specifically how signals from the tumour cells can co-opt the stromal cells to promote tumour progression and decrease therapeutic response. Delineating such signals may lead to development of novel therapeutic strategies.

Pancreatic Ductal Adenocarcinoma (PDAC) has a dismal prognosis with a median survival below six months and an average five-year survival below 5%. This is due to the aggressive nature of the cancer, a lack of effective therapy and late diagnosis. The most frequent occurring genetic mutations have been identified with activating mutations in the oncogene KRAS, inactivation of the tumour suppressor CDKN2A in more than 90% of all tumours and loss of TP53 and SMAD4 function occurring in 50-60% of all cases.

The clinical benefit of chemotherapy is very limited and the abundant stroma is known to confer therapeutic resistance. Critically, PDAC is characterised by an extensive activated stroma and desmoplasia, which also affects tumour growth and metastasis. However, the mechanisms whereby tumour cells recruit and co-opt “normal” stromal cells and conversely how the stromal cells support tumour cell growth and impair drug sensitivity is not well known. Delineating these mechanisms is therefore important and may lead to the identification of novel therapeutic targets in both the tumour and the genetically stable stromal cells.

Analysing signaling through the interrogation of post-translational modifications
To define how tumour and stromal cells signal to each other we utilise state of the art mass spectrometry, which allow us to interrogate the cellular proteome and post translational processes in a robust and quantitative manner. To analyse reciprocal cell communication, we have developed methods for labeling heterotypic cellular populations, which allows the assignment of identified signalling molecules to their cell of origin (Jorgensen et al., Science 2009). As such, we perform direct co-cultures between tumour and stromal cells and analyse how signaling is specifically regulated in the individual cell types. The major advantage of this approach is that all exchanged signals are included in the analysis and as such, heterotypic cell signaling is interrogated as a multivariate system. More recently, we have developed an improved methodology based on cell-specific labeling using precursors of amino acids, which has allowed us to study direct co-cultures for up to 10 days (Tape et al., Mol Cell Proteomics 2014). Because extended co-cultures permit signals to evolve, for example, a signal from cell type A accumulates sufficiently to activate cell type B, which then elicits a reciprocal signal to cell type A, individual signals can be perturbed and the consequences can be determined.  As such, this approach has dramatically improved our ability to interrogate how reciprocal cellular signalling networks develop between tumour and stromal cells. We are currently utilising this technology to map which signals are exchanged between tumour and stromal cells in pancreatic cancer.

In parallel, we have developed phenotypic assays to monitor the effect of tumour-stroma signalling and are currently conducting a loss of function genetic screen to identify components that are required for stromal co-option. Integration of the biochemical signalling network with a functional readout allows us to identify regulated and functionally important signals in a cell specific manner. Recently this has led to the identification of novel regulatory mechanisms whereby tumour cells communicate with stromal cells and we are currently evaluating these as putative therapeutic targets.


Figure 1
: Pancreatic Ductal Adenocarcinoma (PDAC) is characterised by extensive stromal reaction and desmoplasia. Immunohistochemistry for epithelia (pan-cytokeratine), activated fibroblasts (alpha Smooth muscle actin, aSMA) and collagen (Massons Trichrome) shown on pancreatic tissue isolated from Genetic Engineered Mouse model of Pancreatic Cancer. Shown is normal wild type (WT), KRas expressing early stages pancreatic ductal neoplasia (KC) or KRas/P53R172H expressing PDAC. Noteworthy, the epithelia loses its structure progressively as disease develops alongside an extensive fibroblast activation and collagen deposition.

Development of a semi-automated platform for the enrichment of phosphorylated peptides
Protein post-translational modification (PTM) constitutes a major regulatory mechanism of cellular signalling and controls protein stability, activity, subcellular localisation and protein-protein interactions. Protein phosphorylation constitutes a critical PTM, which is underscored by the frequent changes to protein kinase and phosphatase activity in cancer. To study protein phosphorylation as a regulatory mechanism in tumour-stroma signalling, we have developed a robust semi-automated platform for the enrichment of phosphorylated peptides (Tape et al., Anal Chem 2014). This platform allows the simultaneous processing of 96 samples in less than one hour and thus facilitates experiments where multiple conditions can be evaluated at high fidelity. Utilising this platform we have analysed how mutant KRAS alters the cellular signalling network. Although mutant KRAS has been widely studied, there is no comprehensive map of the widespread changes occurring in cells expressing the mutant protein. As such we compared the effect of expressing the frequently occurring mutated KrasG12D across multiple pancreatic cancer cell lines and conditions, allowing us to pinpoint the core regulatory network that is controlled across multiple conditions.

Development of a mass spectrometry assay to quantify kinases
In addition to protein kinase activity, levels of expression and stability are also frequently altered in tumour cells. However, robust quantification of all protein kinases is not easily obtained due to a low level of expression and the lack of reagents (antibodies). To address this we have developed a mass spectrometric assay to accurately quantify human kinases (Worboys et al., Nat Methods 2014). In order to quantify most proteins by mass spectrometry they are enzymatically cleaved into peptides, which then are measured as proxies for the protein level. Importantly, the underlying assumption is that peptides unique to the protein can be used to determine their level of expression. However, for proteins that are subjected to extensive post-translational modifications, such as protein kinases, this may not always hold true. In fact, we observed that indeed it is critical to assess how well each peptide represents the level of the intact protein in order to avoid bias in the measurement. Using this assay, we have quantified the levels of protein kinases across human pancreatic cancer cells to identify putative targets with altered level of expression.

 

Figure 2: Working model of reciprocal tumour-stroma signaling. Expression of mutant KRas in tumour cells leads to changes in their cellular signaling network alongside increased secretion of paracrine acting growth factors, cytokines and morphogens. Neighboring genetically normal stromal cells respond to these changes and consequently adapt an ‘activated’ phenotype, which contribute to the desmoplastic reaction by increased expression of extracellular matrix proteins and growth factors. These changes to the extracellular environment are hypothesised to elicit a subsequent change to the signalling in the tumour cells, leading the increased aggressive behavior and resistance to therapy.