Richard O. Hynes
During 2013, the Hynes laboratory made significant progress on a number of projects under the Ludwig Center program. Our research is focused on dissecting the influences of the tumor microenvironment, both in the primary tumor and during metastatic spread, in each case enhancing the extent of metastasis. Although it is well established that mutations in oncogenes and tumor suppressor genes are involved in establishing primary tumors, there is not, as yet, much evidence for mutations that specifically lead to metastasis. Such mutations may yet be discovered in the wealth of genomic data currently emerging but, at present, it is clear that many other processes than mutations within the tumor genome play key roles in the development, progression and maintenance of metastases. These include multiple influences from cells of the microenvironment (tumor stroma) including direct cell-cell interactions as well as signals by soluble factors and the extracellular matrix. Those are the focus of much of our research. In prior years, we have used both genomic and proteomic screens to identify genes and proteins that are up- or down-regulated during metastasis and we have shown that many of them are causal in regulating metastasis. In our current research supported by the Ludwig Center, we are pursuing these leads in greater depth.
Functions of platelets and leukocytes in enhancing metastasis.
It has been known for a long time that blood platelets enhance metastasis but how they do so has been unclear. Using genetically engineered mice that we had developed, we showed that the adhesion of platelets to tumor cells in the circulation greatly enhances metastasis. We went on to show that platelets do so by their interactions with tumor cells. Tumor cells activate platelets and, in turn, the platelets activate the tumor cells. They do this by binding to them (through the adhesion receptors that we had earlier identified) and by secreting growth factors that stimulate the tumor cells into a more malignant state. We identified the relevant growth factor (TGF-b) and showed that platelets stimulate both the TGF-b signaling pathway and another signaling pathway (NFkB) within the tumor cells and turn them into migratory and invasive cells. We also showed that if we block those two signaling pathways in tumor cells in mice, metastasis is eliminated. That work was published a little over two years ago (Labelle et al, 2011).
We have subsequently followed up to analyze in greater detail this interaction between platelets and tumor cells and have also discovered that the platelets recruit other blood cells to assist in promoting metastasis (Labelle et al, 2012, 2014). We have shown that tumor cells and platelets arrest together at the distant site where a metastasis is beginning to seed and that the platelets then release additional soluble factors (called chemokines) that recruit white blood cells called granulocytes. If we eliminate or mutate the platelets, or block the chemokine signaling pathways, the granulocytes are not recruited. Both the platelets and the granulocytes are essential for the tumor cells to exit from the blood vessel and seed metastases. Later recruitment of another type of blood cell (monocytes) is important for further growth of the metastases. Thus, metastasis involves a complex cooperation between tumor cells and several types of normal cells. This offers the potential for interfering with the signaling between these various cell types as a novel way to inhibit metastasis. We continue to investigate this fascinating process further.
We also investigated possible interactions of platelets with circulating tumor cells and that led to a collaboration with the Wittrup laboratory in the Koch Institute and clinicians at a local hospital on surface antigens of disseminated tumor cells (Yao et al, 2013).
Involvement of integrins (cell surface receptors for proteins of the extracellular matrix) in tumor progression and metastasis.
We have worked for many years on the important interactions between cells and the surrounding network of proteins that make up the extracellular matrix. This forms scaffolds that provide mechanical support for all tissues. These extracellular matrix (ECM) scaffolds provide the substrates for cell adhesion and migration and play essential roles in development of tumors and in tumor angiogenesis (the essential formation of blood vessels to supply the tumors). The ECM also provides vital signals for cell growth and survival and it is clear that ECM signals contribute in important ways to the resistance of tumor cells to chemo- and radio-therapy. The major cellular receptors for ECM proteins are known as integrins and they both bind to the ECM to promote adhesion and migration events important for tumor spread and also transduce signals into the tumor cells providing growth and survival stimuli. Changes in integrins are prevalent in tumors and we have a long-standing program investigating those. This has led us to develop many mutant mice lacking specific integrins in tumor cells and in blood vessels as well as ways to inhibit integrin functions both singly and multiply and we have used those methods to dissect the roles of these important receptors. In previous years we have used these methods to investigate extensively tumor angiogenesis (references not cited here) and, in the past year, we have collaborated with other groups to show the involvement of specific integrins in the seeding of acute myeloid leukemia in the bone marrow (Miller et al, 2013). We also collaborated with other groups in the MIT Ludwig Center (Bhatia, Jacks) in studies of adhesion of metastatic lung cancer cells to ECM (Reticker-Flynn et al, 2012) and those collaborations continue.
Role of the Hippo-YAP signaling pathway in promoting metastasis.
We used the same techniques developed in our integrin work to identify and dissect a novel role for the Hippo-YAP signaling pathway in metastasis. The Hippo kinase cascade transmits signals from integrins and the extracellular matrix and from cell-cell contacts into the cells to inhibit the transcription factor YAP. YAP had previously been shown by others to enhance cell and tissue growth but there was no indication that it played any role in metastasis. We showed that activation of YAP greatly enhanced metastasis of melanomas and breast cancer and demonstrated that it does so by interactions with a set of transcription factors called TEADs (Lamar et al, 2012). During the past year we have defined the genes that are regulated by the YAP/TEAD cooperation and begun to define which of those genes are responsible for the enhancement of metastasis. We are also dissecting how the tumor suppressor, merlin, a linker between cell surface signals and the Hippo/YAP pathway, and the oncogenic kinase SRC regulate YAP activity. Both merlin and SRC are known regulators of metastasis.
Adhesion GPCR surface receptors in metastasis.
In earlier work, we discovered the adhesion GPCR, known as GPR56 as a suppressor of tumor growth and metastasis and showed that it suppresses tumor angiogenesis. That line of research left the lab with a former postdoc, who left to set up her own laboratory. However, we became intrigued by the properties of the entire family of adhesion GPCR receptors (about 3 dozen genes) because of their dual roles as receptors for extracellular proteins and as G-protein-coupled signal transduction receptors. Most of the family were little studied (indeed not known until completion of the genome sequence) but at least one other had been shown to affect metastasis and several had been implicated in regulation of stem cell proliferation. So we conducted a screen of the entire family for up- or down-regulation during metastasis, for associations with cancer prognosis and for mutations detected in the cancer genome projects. These analyses focused our attention on a limited subset of the family as likely contributors to metastasis and we have now shown by overexpression and knockdown experiments in mouse models of breast cancer that at least two of them clearly regulate metastasis and we are further investigating their functions and expect to be able to publish those results in the coming year.
Other genes/proteins mediating connections between external stimuli and intracellular responses during metastasis.
Other proteins discovered in our earlier screens for metastasis enhancers are also under active study in our laboratory. The first of these is the cell surface receptor, CDCP1, which we discovered in a proteomics screen of membrane proteins that are increased on metastatic cells. We showed that this protein is a very effective enhancer of metastasis in melanoma cells and subsequently other groups have implicated it in other cancers. We showed that a single amino acid (tyrosine) in the intracellular domain of CDCP1 is essential for its prometastatic functions. This tyrosine binds to and is phosphorylated by the oncogenic protein SRC and that is key in activating metastasis. CDCP1 also binds and activates other intracellular signaling proteins in tumor cells, reducing cell-cell adhesion and enhancing cell migration and invasion, processes that contribute to metastasis. The external domain of CDCP1 has a structure suggestive of a receptor for extracellular proteins, so we are attempting to identify the external interactors and dissect the roles of the transmembrane signaling involving SRC and other intracellular proteins.
In another earlier screen of genes upregulated in metastasis, we identified RhoC and IQGAP1 – both upregulated in metastatic melanoma cells. We showed conclusively that RhoC is an enhancer of metastasis and published that result some years ago. That conclusion has since been widely replicated by other groups in a wide variety of cancers.
At that time we had some indications that IPGAP1 is also a metastasis enhancer but we did not have enough conclusive evidence to publish the result. Recently, several new results have reinvigorated our interest in pursuing this preliminary evidence. It has been shown that both RhoC and IQGAP1 are concentrated in invadopodia, cell protrusions that enable tumor cells to invade surrounding extracellular matrix. Furthermore, it has been demonstrated that RhoC and IQGAP1 interact. These newer results suggest that our earlier implication of IQGAP1 in invasion and/or metastasis is likely correct and we are working actively on testing this possibility.
We will be continuing all the lines of research discussed above. We expect to publish on several of them in the coming year. Our goals are to establish well defined modulators of different steps in the metastatic cascade – intravasation, arrest in the vasculature, extravasation, initial seeding of metastases and growth and survival of those seeds. The proteins on which we focus are all involved in the interactions with the tumor microenvironment and the transduction of signals between different cells and between extracellular matrix and cells. There is good clinical precedent for effective intervention in such interactions because many of them occur in the extracellular space. Anti-integrin drugs are well established in therapies against thrombosis, inflammation and autoimmune disease and are under active investigation for anti-cancer therapy, GPCRs are the most widely druggable targets in the human genome and, although adhesion GPCRs are not yet clinical targets, we hope that our work may lead in that direction and that our work on the interconnections between SRC, YAP and CDCP1 will open new routes to intervention.
- Labelle, M., Begum, S. and Hynes, R.O. (2011). Direct Signaling Between Platelets and Cancer Cells Induces an EMT-Like Transition and Promotes Metastasis. Cancer Cell 20: 576-590. PMID 22094253 PMC3487108
- Labelle, M. and Hynes, R.O. (2012). The initial hours of metastasis: the importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discov. 2(12):1091-1099. doi: 10.1158/2159-8290.CD-12-0329. Epub 2012 Nov 19. PMID:23166151
- Labelle, M., Begum, S. and Hynes, R.O. (2014). Platelets guide the formation of early metastatic niches. (Ms submitted).
- Yao X, Labelle M, Lamb CR, Dugan JM, Williamson CA, Spencer DR, Christ KR, Keating RO, Lee WD, Paradis GA, Begum S, Hynes RO, Wittrup KD. (2013). Determination of 35 cell surface antigen levels in malignant pleural effusions identifies CD24 as a marker of disseminated tumor cells. Int J Cancer. 2013 Jun 17.
- Miller PG, Al-Shahrour F, Hartwell KA, Chu LP, Järås M, Puram RV, Puissant A, Callahan KP, Ashton J, McConkey ME, Poveromo LP, Cowley GS, Kharas MG, Labelle M, Shterental S, Fujisaki J, Silberstein L, Alexe G, Al-Hajj MA, Shelton CA, Armstrong SA, Root DE, Scadden DT, Hynes RO, Mukherjee S, Stegmaier K, Jordan CT, Ebert BL. (2013). In Vivo RNAi Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling. Cancer Cell. 2013 Jun 11. doi:pii: S1535-6108(13)00197-9. 10.1016/j.ccr.2013.05.004. PMID:23770013
- Lamar, JM, Stern, P., Liu, H., Schindler, JW, Jiang, Z. and Hynes, RO. (2012). The Hippo pathway target, YAP, promotes metastasis through its TEAD interaction domain. Proc Natl Acad Sci U S A. 109(37):E2441-50. doi: 10.1073/pnas.1212021109. Epub 2012 Aug 13. PMID:22891335; PMC3443162