Cells within a tumour can be broadly classified into two types:
1) Cancer cells (mutated).
2) Stromal cells (not mutated).
It's well established that mutations in cancer cells can drive phenotypic changes within cancer cells. For example, a point mutation in a kinase gene can result in hyperactive signalling — causing a cancer cell to grow too fast. When a cell is affected by its own mutation, it's known as a cell-autonomous event.
Most genotype-to-phenotype studies in cancer focus on cell-autonomous events.
But in a real tumour, mutated cancer cells are not alone. They sit alongside healthy, non-mutated stromal cells. In isolation, stromal cells are well behaved. However, in a tumour, stromal cells can be coerced into bad behaviour by cancer cells. They can be bullied by their mutated neighbours.
When a mutation in one cell regulates the behaviour of a neighbouring cell, this is called a non-cell-autonomous event.
Despite its huge role in cancer, non-cell-autonomous signalling is poorly understood. Most examples are anecdotal to a single pathway or molecule. We actually know very little about how mutations signal beyond cancer cells. It's often presumed to occur, but we have no idea how much, and what the consequences are.
Why Bully?
Several years ago I started working on how mutations signal beyond cancer cells in pancreatic ductal adenocarcinomas (PDA).
PDA is a nasty disease that contain lots of stromal cells. In fact, some tumours contain even more stromal cells than cancer cells.
PDA is driven by mutations in a gene called KRAS. I wanted to understand how mutant KRAS signals through PDA cancer cells and stromal cells. If we can understand this bullying process better, we might be able to offer new treatments to patients.
I started by measuring how KRAS affects cell-autonomous signalling. Using high-throughput multivariate phosphoproteomics, we found that KRAS regulates a very specific section of cancer cell signalling. Mainly MAPK, CDK1/5, and CKII pathways.
Next we looked at non-cell-autonomous KRAS signalling. It's been known for a while that KRAS communicates with stromal cells via a protein called sonic hedgehog (SHH) (no not that one). We found the same thing.
But we also noticed something else: non-cell-autonomous KRAS causes stromal cells to make new, unique growth signals. These signals are distinct to the stromal cells and are not produced by cancer cells on their own. Cancer cell KRAS bullies stromal cells into making new growth signals.
This posited an interesting question: do bullied stromal cells send signals back to the cancer cells? Does KRAS drive reciprocal signalling?
It's easy to hypothesise reciprocal signalling. It's much harder to actually test it. To my knowledge, no one has ever shown mutations can uniquely regulate cancer cells via stromal reciprocal signalling.
Why? Because it's tricky to see. To observe reciprocal signalling, you need the following:
1) You need to measure cell-autonomous, non-cell-autonomous, and a hypothetical reciprocal signalling state in a single experiment. For this, you need a technique that can measure several phosphoproteomes at once.
and....
2) You need to know which signal comes from which cell type. That is, you need a technique that provides cell-specific resolution of two heterotypic phosphoproteomes from multiple conditions.
It's not trivial.
Heterocellular Multivariate Phosphoproteomics
Over the past 4 years I've been developing techniques for multivariate phosphoproteomics and cell-specific isotopic proteome labelling (alongside the CRUK MI Systems Oncology team). I combined these techniques to perform 'heterocellular multivariate phosphoproteomics' (HMP). This technique allowed us to monitor thousands of phosphosites across 10 conditions in two cells at the same time. Just what we needed to test reciprocal signalling.
So what did we find?
When cancer cells non-cell-autonomously communicate with stromal cells, the number of regulated phoshosites in cancer cells doubled relative to cell-autonomous signalling. Cancer cells were getting a big reciprocal signal back from the stromal cells.
Reciprocal KRAS activated major signalling hubs (such as AKT) - regulating transcription, protein abundance, metabolism, cell death, and cancer cell growth. KRAS uniquely regulates cancer cells by bullying its neighbours.
We put together a little video describing KRAS reciprocal signalling:
Reciprocal signalling is not non-cell-autonomous signalling in reverse. A reciprocal axis starts with an oncogenic mutation, travels through a differentiated stromal cell, and then – using new signals produced by stromal cells – returns to activate pathways the cancer cell cannot activate on its own.
Reciprocal signalling allows mutations to expand their signalling capacity via differentiated heterocellularity.
This work has just been published over at Cell. If you're into spectra, we've also made all the raw mass-spec data available at PRIDE (PXD003223).