Program and Contributions to Science
Movie shows the process of early dissemination in real time from premalignant lesions using intra-vital high resolution microscopy. Blue early cancer cells – Red, blood vessels Nature. (2016) Dec 14 PMID:27974798.
(i) Mechanisms of early dissemination, dormancy and metastasis. Why cancer undergoes prolonged phases of stable disease after surgery and/or treatment to then reactivate and progress to metastasis is a key unanswered question in cancer biology. Further, understanding the source of DCCs, how they undergo dormancy and how they still carry metastasis initiating potential is central to preventing metastasis-related deaths. At what time during cancer evolution dormant DCCs appear and how they affect the timing and heterogeneity of metastasis, is also poorly studied. It is known that DCCs detected in patients before the manifestation of breast cancer metastasis contain fewer genetic abnormalities than primary tumors or than DCCs from patients with metastases. These findings and those in pancreatic cancer and melanoma models argued that dissemination might occur during early stages of tumor evolution. Yet, the mechanisms that might allow early-disseminated cancer cells (eDCC) to complete all steps of metastasis were unknown. Our work in our early dissemination program shows that in early lesions (EL), before any overt primary tumor (PT) masses are detected, there is a sub-population of Her2+/P-p38lo/P-ATF2lo/TWISThi/E-cadherinlo early cancer cells that are invasive and disseminate to target organs. Intra-vital imaging (see movie) and organoid studies of early lesions revealed that Her2+ eDCC precursors locally invaded, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent EMT-like dissemination program but without complete loss of epithelial phenotype that was reversed by Her2 or Wnt inhibition. Surprisingly, while the majority of eDCCs are TWISThi/E-cadherinlo and dormant, they eventually initiate metastasis.We also revealed how the HER2 oncogene activates through CCL2 signaling the recruitment of tissue resident macrophages that help early DCCs to enter circulation. Targeting these macrophages early in cancer evolution reduced metastasis late in cancer progression. Impact: Our work identifies a mechanism for early dissemination whereby Her2 aberrantly activates a program similar to mammary ductal branching that spawns eDCCs capable of forming metastasis after a dormancy phase. Our findings change our understanding of how certain oncogenes may initiate dissemination before triggering aggressive proliferation and how tumor suppressor pathways might suppress metastasis. We may also be able to understand how eDCCs found metastasis directly and/or through the preparation of eDCC-orchestrated pre-metastatic niches for later arriving DCCs to colonize target organs. Our work is also revealing how macrophages might contribute to the regulation of early dissemination and late metastasis. Our program also focuses on identifying markers that might pinpoint early DCCs vs late DCCs and that may allow selectively targeting these cells. References: Harper KL, et al. Mechanism of early dissemination and metastasis in HER2+ mammary cancer. Nature. (2016) Dec 14. doi: 10.1038/nature20609. [Epub ahead of print] PMID:27974798. Hosseini, H, et al. Early dissemination seeds metastasis in breast cancer. Nature. (2016) Dec 14. doi: 10.1038/nature20785. [Epub ahead of print] PMID:27974799. Linde and Casanova-Acebes et al., Nat Commun (2018).doi: 10.1038/s41467-017-02481-5
(ii) Mechanisms of disseminated tumor cell dormancy and metastasis. We explored why in many patients metastasis remain dormant in some organs like the bone marrow, but grow in others like lungs. We found that the bone marrow contains high levels of TGFβ2, which induced dormancy via TGFb-RI/RII/RIII complexes and p38. In the lungs, where TGFβ2 is much less abundant dormancy is short-lived and DTCs rapidly form metastasis. In contrast, these signals trigger a long-lived program of quiescence in the bone marrow. Analysis of the transcriptional and epigenetic mechanisms active in DTCs that enter dormancy pinpointed retinoic acid as a micro-environmental pro-dormancy cue. We found that the orphan nuclear receptor , which regulates lineage commitment and is silenced in human tumors, is spontaneously upregulated in solitary dormant tumor cells. Retinoic acid induces TGFb2, NR2F1 expression and dormancy of DTCs that acquire a highly repressive chromatin state. Surprisingly, NR2F1 induces NANOG to induce dormancy in bone marrow DTCs. This revealed that dormant tumor cells resemble adult stem cells. We found that a transient treatment with low dose of 5-azacytidine, followed by all–trans retinoic acid, restored the NR2F1-driven program and long-term in vivo quiescence of previously malignant cells. Validating our work we found that the dormancy mechanisms and markers predict for longer metastasis-free periods in ER+ breast cancer patients and these signatures can be found in single DTCs from prostate cancer patients in clinical dormancy for up to 18 years and not in DTCs from patients with advanced disease.
– Hypoxia as a DTC dormancy programmer. We also discovered (Fluegen et al., Nat Cell Bio, 2017) that hypoxic primary tumor (PT) microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia genes (GLUT1, HIF1a). Mechanistic analysis revealed that post-hypoxic DTCs were frequently NR2F1hi/DEC2hi/p27hi/TGFb2hi and dormant. NR2F1 and HIF1a were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1hi expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER- breast cancer cells, post-hypoxic ER+ breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that PT hypoxic microenvironments give rise to a sub-population of dormant DTCs that evade therapy and may be the source of disease relapse and poor prognosis associated with hypoxia.
Impact: We revealed 1- a new “seed and soil” mechanism that regulates DTC dormancy, 2- novel markers to determine if DTCs may be dormant in patients, 3- atRA and NR2F1 signaling as epigenetic regulators of dormancy and 5- a therapeutic strategy using available drugs to prevent metastasis by inducing dormancy. Based on these findings we have designed a clinical trial using AZA and atRA after hormonal ablation in PCa and identified an agonist of NR2F1 to induce and maintain dormancy via dormancy induction. References: Fluegen G, et al., (2017). Phenotypic heterogeneity of disseminated tumor cells is preset by primary tumor hypoxic microenvironments. Nature Cell Biology (2017) doi:10.1038/ncb3465. Sosa, M.S., et al., (2015). NR2F1 controls tumour cell dormancy via SOX9- and RARb-driven quiescence programmes. Nat Commun 6, 6170. Chéry, L., et al. (2014). Characterization of single disseminated prostate cancer cells reveals tumor cell heterogeneity and identifies dormancy associated pathways. Oncotarget, 1949-2553. Bragado, P., et al., (2013). TGF-b2 dictates disseminated tumour cell fate in target organs through TGF-beta-RIII and p38a/b signalling. Nat Cell Biol 15, 1351-1361. Kim, R.S., et al., (2012). Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer. PloS one 7, e35569.
(iii) Protease receptor and integrin signaling in cancer – the discovery of a molecular mechanism to induce cancer dormancy. In the late 90’s the mechanisms that might regulate dormancy were unclear and not a “hot” topic. Within the paradigm of cancer invasion, the urokinase type plasminogen activator (uPA) and its receptor uPAR were thought to regulate ECM turnover. Our work went on to show that in cancer cells uPA has a signaling function through its GPI-anchored receptor uPAR, which is independent of protease activity and results in robust activation of the ERK1/2 pathway. Because uPAR is GPI anchored it became critical to understand how uPAR can transduce signals. We showed that uPAR interacts via its domain III with α5β1 integrins by binding in cis to the α5 subunit. This interaction activates the integrin and its efficient binding and polymerization of fibronectin into fibrils. Active a5b1 integrins recruited the focal adhesion kinase (FAK), which then recruited the EGFR to activate robust MEK-ERK1/2 signaling. Blockade of uPAR signaling resulted in the disassembly of the above-mentioned complex with a reduction of MEK-ERK1/2 signaling and tumor growth inhibition. This resulted in the reprogramming of tumor cells into long term G0/G1 arrest (quiescence) and a protracted dormancy state. Interestingly, blockade of the uPAR-integrin complex resulted in upregulation of p38 signaling, which was required to further inhibit ERK1/2 signaling and maintain dormancy. Impact: We discovered 1- a reciprocal crosstalk between tumor cells and the microenvironment regulates cancer cell dormancy 2- a novel signaling function for uPAR in cancer cell growth which turned out to be the first molecular mechanism of cancer dormancy, 3- that dormancy induction required not only reduced mitogenic signaling but also p38 stress signaling. These findings led to the development of uPAR blockade strategies with small molecules and biological, some in clinical trials. References: Aguirre Ghiso, J.A. (2002). Inhibition of FAK signaling activated by urokinase receptor induces dormancy in human carcinoma cells in vivo. Oncogene 21, 2513-2524. Aguirre Ghiso, J.A. et al., (1999). Tumor dormancy induced by downregulation of urokinase receptor in human carcinoma involves integrin and MAPK signaling. JCB 147, 89-104. Aguirre-Ghiso, J.A., et al., (2001). Urokinase receptor and fibronectin regulate the ERK(MAPK) to p38(MAPK) activity ratios that determine carcinoma cell proliferation or dormancy in vivo. MBoC 12, 863-879. Liu, D., Aguirre Ghiso, J.A., et al., (2002). EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer Cell 1, 445-457.
(iv) The balance between mitogenic and stress signaling as a determinant of dormancy. We focused on the gene programs regulated by the balance between ERK and p38. We found that the ERK to p38 ratio was predictive of cancer cell dormancy or reactivation across different cancers, including HNSCC, breast and prostate. We found that the small GTPase CdC42 was responsible for p38 activation after disruption of the uPAR complex and using a pathway reporter system for monitoring ERK mitogenic and p38 stress signaling in vivo, we discovered that all cancer cells activate p38 upon dissemination but those that go on to metastasize from DTCs silence p38 signaling. When we characterized the survival and quiescence signals downstream of the ERK/p38 ratio we found that p38 induced in dormant cells an unfolded protein response, upregulation of the chaperone BiP and activation of the ER kinase PERK. These pathways contributed to basal dormant cancer cell survival and survival to DNA damaging agents. We also showed that p38 activated the transcription factor ATF6 to induce Rheb and an alternative mTOR pathway activation that conferred survival signals for dormant (G0/G1 arrested) cancer cells. Finally, we revealed a transcription factor network regulated by ERK/p38 balance required for tumor cell quiescence during dormancy. Impact: These studies were the first to provide mechanistic understanding on how malignant cancer cells can reprogram into dormancy. We also defined key signaling and transcriptional mechanisms that contributed to the G0/G1 arrest and survival of malignant cells reprogrammed into dormancy. References: Aguirre-Ghiso, J.A., et al., (2004). Green fluorescent protein tagging of extracellular signal-regulated kinase and p38 pathways reveals novel dynamics of pathway activation during primary and metastatic growth. Cancer Res 64, 7336-7345. Ranganathan, et al., (2006). Functional coupling of p38-induced up-regulation of BiP and activation of RNA-dependent protein kinase-like endoplasmic reticulum kinase to drug resistance of dormant carcinoma cells. Cancer Res 66, 1702-1711. Schewe, D.M., and Aguirre-Ghiso, J.A. (2008). ATF6alpha-Rheb-mTOR signaling promotes survival of dormant tumor cells in vivo. Proc Natl Acad Sci USA 105, 10519-10524. Adam, A.P. et al., (2009). Computational identification of a p38SAPK-regulated transcription factor network required for tumor cell quiescence. Cancer Res 69, 5664-5672.
(v) Adhesion, stress signaling and autophagy. We found that p38 is activated upon loss of b-integrin adhesion signaling leading to ERK1/2 inhibition and induction of the pro-apoptotic protein BimEL. This is key to induce anoikis, proper development of the mammary tree. We found that the HER2 oncogene inhibits p38 and this accelerated cancer progression. We also discovered that proper adhesion signaling limits activation of the endoplasmic reticulum PERK, which limits mammary cancer initiation by blocking proliferation. We found that ErbB2 signaling is dependent on optimal activation of eIF2a signaling and that causing an imbalance in P-eIF2a levels killed ErbB2-overexpresing cells. We also showed that PERK-eIF2a-ATF4-CHOP activation in ECM-detached mammary epithelial cells induces autophagy and antioxidant responses for survival. Aut
ophagy was also quickly activated by PERK-dependent activation of an LKB1-AMPK-TSC2 pathway that blocked mTOR activation and cancer cells co-opt PERK signaling for survival. We also published that in multiple myeloma, modulation of the UPR and quiescence pathways allows cancer cells that survive bortezomib treatment to persist in a dormant but still stress resistant phenotype that may propel recurrences. Impact: We revealed that PERK has a dual role as tumor suppressor by performing antioxidant functions in normal cells, but it can also be co-opted to optimize stress signaling in oncogene expressing cells. The impact of our UPR program led to a research collaboration with Eli Lilly to test UPR inhibitors to target this pathway in several cancer types. References: Schewe DM and Aguirre-Ghiso, JA (2009). Inhibition of eIF2a dephosphorylation maximizes bortezomib efficiency and eliminates quiescent multiple myeloma cells surviving proteasome inhibitor therapy. Cancer Res. Feb 15;69(4):1545-52. Avivar-Valderas, A., et al., (2011). PERK integrates autophagy and oxidative stress responses to promote survival during extracellular matrix detachment. Mol Cell Biol 31, 3616-3629. Wen, H.C., et al., (2011). p38 Signaling Induces Anoikis and Lumen Formation During Mammary Morphogenesis. Science Signaling 4, ra34. Avivar-Valderas A et al., (2013). Regulation of autophagy during ECM detachment is linked to a selective inhibition of mTORC1 by PERK. Oncogene, 2013 Oct 10;32(41):4932-40
(vi) Integrating dormancy in the paradigm of metastasis. We have placed important effort in integrating the current biology of cancer dormancy and stress signaling in the paradigm of cancer progression. Through a series of comprehensive reviews, commentaries and perspectives we have outlined the evolution of the cancer dormancy field and its integration with metastasis mechanisms. This body of work and our research has been highly cited in academic and public venues and has influenced the development of dormancy programs. References: Aguirre-Ghiso, J.A. (2007). Models, mechanisms and clinical evidence for cancer dormancy. Nature Reviews Cancer 7, 834-846. Aguirre-Ghiso, J.A., et al. (2013). Metastasis awakening: targeting dormant cancer. Nat Med 19, 276-277. Sosa, M.S., Bragado, P., and Aguirre-Ghiso, J.A. (2014). Mechanisms of disseminated cancer cell dormancy: an awakening field. Nature Reviews Cancer 14, 611-622.
Complete list of publications: http://www.ncbi.nlm.nih.gov/pubmed/?term=aguirre-ghiso
>15,500 citations, Google Scholar. h-index 43, i10-index 70.