Investigación 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) Early dissemination and early DCC dormancy. Our lab has discovered that dormant breast cancer DCCs and metastasis can originate very early during cancer evolution, disseminating during pre-malignant stages and aided by innate immune cells. We identified a mechanism for early dissemination whereby Her2 aberrantly activates a program similar to mammary ductal branching that spawns early DCCs (eDCCs) capable of forming metastasis after a dormancy phase. We also revealed how the HER2 oncogene activates through CCL2 signaling the recruitment of tissue resident macrophages that help eDCCs to enter circulation. Targeting these macrophages early in cancer evolution reduced metastasis late in cancer progression. Together, we try to understand how eDCCs orchestrate signals intrinsic to cancer cells and those from the microenvironment to remain dormant and persistent and how alterations of these signals lead to reactivation into outgrowing metastases. We also aim to identify markers that might pinpoint early DCCs vs. late DCCs and may allow selectively targeting these cells. To this end we recently completed a full single cell sequencing study of early and late primary lesions as well as DCCs from matched early and late lungs identifying the transcriptional programs that drive early DCC dormancy. Understanding mechanisms of disseminated cancer cell (DCC) plasticity, represented by the transitions between epithelial and mesenchymal cell fates (EMT and MET), has been shown to closely associate with tumor cell proliferation, cancer dormancy, and metastasis. Our early DCC studies have identified the pluripotency regulator ZNF281 (aka Zfp281, ZBP99, GZP1) in controlling anterior-posterior axis formation and epiblast maturation through direct transcriptional and epigenetic regulation of Nodal/TGFb signaling pathway, which is well known for its roles in EMT and cancer metastasis as well as in BCa stemness. ZNF281 induces a permissive state for heterogeneous mesenchymal-like (M-like) transcriptional programs. These programs carry a dormancy signature and are absent in proliferative primary tumors and metastasis. Harper, K, Sosa MS., et al., (2016) Nature. PMID 27974798 Hosseini et al., (2016) Nature. PMID 27974799 Linde and Casanova-Acebes et al., (2018) Nat Commun. PMID 29295986 Nobre et al., (2022) Nat Cancer. PMID: 36050483 (ii) Stem cell and niche biology in cancer dormancy. We have explored why in many patients DCCs persist and remain dormant in the bone marrow and other organs. We found that the bone marrow contains high levels of TGFβ2, which induced dormancy via TGFβ-RI/RII/RIII complexes and p38 signaling. We used mouse genetics to show that a key source of TGFβ2 is the NG2+/Nestin+ mesenchymal stem cells (MSCs) that induce hematopoietic stem cell dormancy and self-renewal and also proved that these MSCs induce and maintain dormancy of breast cancer DCCs in the bone marrow. We also determined that the retinoic acid signaling via the nuclear receptor NR2F1 is required for survival of HNSCC DCCs in the bone marrow but it controls dormancy of these same DCCs in the spleen and lungs. Interestingly the expression of NR2F1 can be initiated in the primary site by a combination of hypoxic signals and the presence of specialized macrophages that coordinate the induction of NR2F1 and SOX9 and prime intravasating DCCs to enter dormancy post extravasation. We have also discovered that dormant tumor cells assemble their own pro-dormancy niche by coordinating the above signals with the upregulation if collagen-III and the assembly of curly collagen matrices that sustain dormancy via DDR1 signaling STAT1 signaling. We have also explored how in lung cancer niches orchestrated by tissue resident macrophages (TRMs) and how these are altered by early lung cancer cells and later recruited bone marrow derived macrophages regulate lung cancer growth initiation. This work revealed that TRMs orchestrate a tissue regenerative and immune evasive program that enables lung cancer initial growth. Together these studies have provided a molecular description of how a reciprocal interaction between DCCs and their niches are essential for survival and dormancy of DCCs across different cancers. Bragado, P., et al.,(2013). Nat. Cell Bio. PMID 24161934 Sosa MS et al. (2015) Nat Commun. PMID 25636082 Nobre AR et al., (2021) Nat Cancer. PMID: 34812843 Di Martino Jet al., (2021) Nat Cancer. PMID: 35121989 Casanova-Acebes, M., et al., (2021) Nature. PMID: 34135508 Borriello L. et al., (2022) Nat Commun. PMID: 35110548 (iii) Mechanism of uveal melanoma dormancy. The primary concern in uveal melanoma (UM) patient is the development of liver metastasis after primary tumor resection and the lack of effective treatment to treat the same. UM DCCs can leave the primary tumor at very early stages of progression as observed in other types of cancers, seed target organs and remain dormant for years, until some change “awakens” DCCs causing metastasis after 10-15 years. We hypothesize that dormant DCCs enter quiescence and cannot be eradicated by anti-proliferative therapies. We further hypothesize that these mechanisms could be targeted to prevent awakening and metastasis, as well as to identify biomarkers that could be used to predict dormant disease in patients and therapy response. This approach has not been attempted in uveal melanoma. The primary aim of this effort in the lab is to provide the preclinical basis for new treatment options for early as well as late-stage UM patients. TGFβ2 signaling was found to be implicated in the dormancy of HNSCC DCCs in the bone marrow, demonstrated as being a ‘restrictive soil’ compared to the lungs for example. Interestingly, liver cells also express TGFβ2. Thus, we hypothesize that TGFβ2 could be maintaining UM DCCs dormancy in the liver for years until age-related changes in the liver ME induces their awakening. The lab is also currently exploring this aspect of dormancy-maintaining signaling as well as optimizing targeted therapies as a possible therapy option for UM patients. Kadamb and Lopez-Anton et al., (2022) In Preparation The et al., (2020) Mol Cancer Ther PMID: 32430489 Lapadula et al., (2019) Mol Cancer Res PMID: 30567972 (iv) Tissue homeostasis, aging, and dormancy: We are dissecting out the interaction between tissue resident host cell populations in the metastatic niche and DCCs, and how this may regulate dormancy and re-awakening. Once DCCs arrive at distal sites they are exposed to niche-specific homeostatic processes. These processes are largely driven by stromal and immune cell populations. Given that dormancy is a long-lived process that can span many years, we hypothesize that these homeostatic processes likely induce dormancy, and that the loss of tissue homeostasis results in DCC re-awakening. Over the span of an organism’s life, many age-related changes result in a loss of homeostasis which have been shown to affect DCC re-awakening. Age-induced modifications in pulmonary fibroblast WNT-signaling induces a dormancy-to-reactivation switch in melanoma DCCs resulting in efficient metastatic outgrowth in aged lungs. We are also beginning to investigate how age-related changes in the immune and stromal compartment may influence dormancy and re-awakening at distal sites and how specific factors that reverse aging phenotypes may affect the fate of DCCs and their switching from dormancy to reactivation in melanoma and breast cancer. Fane et al., (2022) Nature. PMID: 35650435 (v) Epigenetic programs of dormancy and therapeutic targeting. Targeting dormancy mechanisms for metastasis suppression: Analysis of the transcriptional and epigenetic mechanisms active in dormant DCCs pinpointed retinoic acid as a micro-environmental pro-dormancy cue. We found that the orphan nuclear receptor NR2F1, which regulates lineage commitment and is silenced in human tumors, is spontaneously upregulated in solitary dormant tumor cells. Retinoic acid induces TGFβ2, NR2F1 expression, and dormancy of DCCs. We found that a transient treatment with a low dose of 5-azacytidine (AZA), followed by all-trans retinoic acid (atRA), restored the NR2F1-driven program and long-term in vivo quiescence of previously malignant cells. We showed that NR2F1-independent pathways induced in parallel upon reprogramming also play an important role in dormancy induction and they depend on TGF-SMAD4 signaling. This mechanism is due to specific remodeling of enhancers linked to TGFb-SMAD4 signaling. The AZA+atRA strategy is currently being used in a clinical trial in prostate cancer. Our epigenetics work has also revealed that in melanoma mutations in the chromatin regulator ARID2 acquire a mesenchymal slow cycling buyt highly disseminating phenotype. Further analysis of epigenetic pathways focused on the histone variants macroH2A, especially the isoform macroH2A2, and we found this histone variant enforces a stable dormant phenotype in DCCs by activating dormancy and senescence genes that limit metastasis initiation. We showed that TGF2 and p38a also upregulate macroH2A and thus induce DCC dormancy. Recently, we capitalized on this knowledge and discovered an agonist of the NR2F1 that specifically activates dormancy programs or neural crest stem cell commitment that drives stably malignant cells into a persistent growth arrest in target organs. In addition, we discovered long ago that dormant cells depend on the unfolded protein response for survival and specifically on the PERK pathways. We have now identified an inhibitor the PERK pathway, which is required for the survival of dormant DCCs and when used in vivo blocks metastasis by eradicating dormant DCCs during quiescence. This inhibitor is currently in clinical trials. Ranganathan et al., (2006/2008) Cancer Res. PMIDs, 16452230; 18451152 Sosa, M.S., et al.,(2015). Nat Commun PMID 25636082 Singh D.K. et al., (2021). bioRxiv https://doi.org/10.1101/2021.08.01.454684 Sun D. et. al., (2021). bioRxiv https://doi.org/10.1101/2021.12.07.471619 Khalil B.D. et al, (2021). J. Exp. Med. PMID 34812843 Calvo, V., et al., (2021). bioRxiv https://doi.org/10.1101/2021.07.30.454473 Carcamo S., et a., (2022) Cell Reports. PMID 35385731 (vi) 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 NR2F1 hi/DEC2hi/p27hi/TGFb2hi and dormant. NR2F1 and largecoverHIF1a 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. (vii) 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. JCB 1999Blockade 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 MAPKJCB 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. From Aguirre-Ghiso et al., 2001, MoBC (viii) 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. From Wen et al., Sci Signal, 2011 (ix) 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 (x) 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. From Aguirre-Ghiso et al., Nat Med 2013
