Case Study 4: Membrane trafficking – a key player in metastasis

Summary

• First use of genetically engineered mouse models (GEMMs) to demonstrate roles for endosomal trafficking in metastasis and metastatic niche priming

• New signalling mechanisms linking endocytosis to invasive behaviour

• A novel paradigm for establishment and sensing of chemokinetic gradients that drive invasiveness

We have used GEMMs to demonstrate that membrane trafficking events occurring in PDAC cells drive metastasis in vivo. Amongst these events are: 1) recycling of EphA2 and LPAR1 controlled by Rab-coupling protein (RCP) and N-WASP, respectively; 2) nuclear-capture of endosomes leading to activation of the SRF transcription factor; and 3) release of podocalyxin-containing exosomes, which influence ECM deposition in metastatic target organs.

Work conducted at the CRUK Beatson Institute has made key contributions to our understanding of how endosomal trafficking of receptors is influenced by oncogenes and how that affects cancer cell adhesion and migration. Nevertheless, prior to 2017, it was unknown whether oncogene-driven membrane trafficking influenced metastasis in vivo. We, therefore, instigated studies that have demonstrated roles for specific membrane trafficking events in mutant p53-driven metastasis and metastatic niche priming in vivo. Moreover, development of conditional knockouts for endosomal regulators, and the study of these in mutant p53-driven mouse models of metastatic cancer, has further allowed us to identify new and unprecedented signalling mechanisms linking membrane trafficking to invasive migration and chemotaxis.

Many signalling and adhesion receptors are continuously internalised, trafficking through the endosomal pathway and are then recycled to the cell surface, and the way in which this membrane trafficking occurs is known to affect their function. For example, endocytosis and recycling of receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs) and integrins influences cell migration and invasion. Moreover, we have made major contributions to understanding how oncogenic landscapes drive cancer cell migration by re-programming receptor recycling, which trafficking and cytoskeletal regulators mediate this, and how this ultimately affects cell migration and invasion in vitro. Specifically, in 2009 we showed that mutant p53’s pro-invasive gain-of-function is mediated by its ability to upregulate recycling of RTKs and integrins via a pathway controlled by the trafficking regulator, Rab-coupling protein (RCP) (1). We subsequently elucidated the role of numerous other factors in trafficking events that control cell migration. For instance, we discovered how regulators of the actin cytoskeleton, such as WASH and N-WASP control recycling of integrins and other adhesion receptors to influence invasive cell movement (2,3).

The metastatic cascade is complex, and many processes are necessary for the dissemination of tumour cells from the primary site and their seeding and growth in distant organs. The acquisition of tumour cell invasiveness is one of the processes allowing tumour cells to escape from the primary site but, following this, disseminated tumour cells must survive the rigours of the circulation and lymphatics, then they must successfully exit the vasculature in other organs and adapt to these sites to successfully seed metastases. To recapitulate the events of this metastatic cascade in their appropriate biological contexts we have generated and deployed a battery of mouse models of metastatic cancer. Most pertinently we have generated mouse models of pancreatic adenocarcinoma (PDAC) that recapitulate the ability of mutant p53 to drive metastasis to the liver and lungs – the two most common sites for PDAC to colonise in humans (4). We have exploited this unparalleled expertise in disease-positioned mouse models of metastatic cancer to define how membrane trafficking and cytoskeletal dynamics influence metastasis and to reveal new mechanisms leading to the dissemination of cancer cells and their seeding of distant organs.

Trafficking of EphA2 is required for invasion and metastasis in PDAC

By generating a series of mice with floxed alleles for RabGTPase effectors, RTKs and integrins and crossing these with the mutant p53-driven ‘KPC’ model of PDAC, the Norman, Morton and Sansom laboratories have shown that RCP-dependent recycling of an RTK (EphA2) is critical for metastasis to the lungs and liver but does not influence primary tumour growth (5). This in vivo study then informed the generation of key reagents that allowed us to reveal an unprecedented new signalling pathway through which RTKs communicate with the transcriptional machinery in the nucleus. Indeed, by combining a detailed proteomic and cell biological analysis of cells from EphA2 knockout PDAC, the Norman and Zanivan groups showed that endosomes are captured on the nuclear surface by direct interaction of EphA2’s cytoplasmic tail with the nuclear pore. Guided by a key collaboration with our computational biology unit, we demonstrated that this event alters nucleocytoplasmic flux of monomeric actin to re-programme transcription and evoke strongly invasive phenotypes (6). This allowed development of a model predicting how signalling events occurring close to the nucleus can influence nucleocytoplasmic shuttling and signal to transcriptional machinery. We anticipate that our understanding of this novel nuclear-capture signalling pathway will provide opportunities for targeting invasive PDAC with agents that need to be delivered to the nucleus, such as antisense oligonucleotide technologies.

Trafficking of LPAR1 enables PDAC cells to sense self-generated chemotactic gradients on the road to metastasis

Our long-standing interest in mapping how membrane trafficking is controlled by the actin cytoskeleton encouraged us to generate a series of mice with floxed alleles for actin regulators. Using these mice, the Machesky, Insall and Norman laboratories collaborated to show that tumour-specific knockout of N-WASP, a key actin regulator, does not influence growth of primary PDAC, but reduces tumour invasiveness and metastasis to the liver (7). In parallel, we combined state-of-the-art bioengineering with computational modelling to demonstrate a completely new paradigm for cell migration in which cancer cells generate their own gradients of signalling molecules, such as lysophosphatidic acid (LPA), which promote migration of cells away from the tumour mass to drive dissemination (8). Importantly, our transgenic mice enabled us to determine that N-WASP knockout PDAC cells cannot respond to self-generated gradients of LPA. A detailed mechanistic analysis indicated that knockout of N-WASP reduces endosomal trafficking of the LPA receptor (LPAR1), and for this reason N-WASP knockout cells are unable to respond to the chemotactic gradients of LPA that would normally drive tumour egress (7).

‘Paired’ metastatic and non-metastatic models of PDAC reveal key roles for membrane trafficking in metastatic niche priming

One focus we have is on understanding how primary tumours prime organs for metastasis, how this priming may be assessed and whether the ‘primed’ metastatic niche may be targeted therapeutically. We have exploited our ‘paired’ metastatic (KPC; mutant p53-driven) and non-metastatic (KflC; driven by p53 loss) models of PDAC – previously developed by the Morton and Sansom groups (4) - to study metastatic niche priming. Using this approach, the Norman, Zanivan, Morton, Carlin and Blyth groups, have shown that mice bearing mutant p53-expressing PDAC display alterations in the extracellular matrix (ECM) and pro-invasive microenvironment of the lungs that precede seeding of metastases (9). This inter-organ communication event is mediated by the ability of mutant p53 to influence the sorting of podocalyxin into exosomes released from the primary tumour. Moreover, these exosomes then alter ECM deposition in the perivascular space and the parenchyma of the lungs by altering integrin trafficking in lung fibroblasts. These findings are being actively pursued by the Carlin, Norman and Roberts labs, who are using our paired metastatic/non-metastatic models of PDAC to determine how exosome-mediated alterations to the lung ECM are linked to pre-metastatic recruitment of neutrophils and the generation of immune-suppressive microenvironments. We anticipate that the ability to predict lung metastasis in PDAC will be invaluable to informing the management of the disease.

References

1. Muller PA et al. Mutant p53 drives invasion by promoting integrin recycling. Cell. 2009; 139: 1327-41
2. Yu X et al. N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods. J Cell Biol. 2012; 199: 527-44
3. Zech T et al. The Arp2/3 activator WASH regulates alpha5beta1-integrin-mediated invasive migration. J Cell Sci. 2011; 124: 3753-9
4. Timpson P et al. Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53. Cancer Res. 2011; 71: 747-57 
5. Gundry C et al. Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion. Nat Commun. 2017; 8: 14646
6. Marco S et al. Nuclear-capture of endosomes depletes nuclear G-actin to promote SRF/MRTF activation and cancer cell invasion. Nat Commun. 2021; 12: 6829
7. Juin A et al. N-WASP control of LPAR1 trafficking establishes response to self-generated LPA gradients to promote pancreatic cancer cell metastasis. Dev Cell. 2019; 51: 431-45 e437
8. Tweedy L et al. Seeing around corners: Cells solve mazes and respond at a distance using attractant breakdown. Science. 2020; 369: eabf9141
9. Novo D et al. Mutant p53s generate pro-invasive niches by influencing exosome podocalyxin levels. Nat Commun. 2018; 9: 5069