Abstract SY17-02: Mechanisms driving dormancy of disseminated tumor cells
Authors: Aguirre-Ghiso Julio A. , Sosa Maria Soledad , Bragado Paloma , Avivar-Valderas Alvaro , Estrada Yeriel , Adomako Alfred , Harper Kathryn , Ekpin Esther 
Published: August 2012

Abstract
The vast majority of cancer related deaths are due to metastasis. However, our understanding of this process is limited. This is clearer when we consider that these lesions can develop years or decades after successful primary tumor treatment. These long periods of clinical remission can be explained by minimal residual disease (i.e. Disseminated Tumor Cells, DTCs) entering a non-productive or dormant state. However, our limited knowledge of these events has curtailed the development of strategies to prevent metastasis. Key questions in this field are: How does early dissemination start and what are the mechanisms? How does the tumor microenvironment aid this process? Are primary tumor (PT) niches responsible for DTCs growth or quiescence? What role does the microenvironment in the metastatic niche play in determining the timing of DTC dormancy?
In patients, DTCs that are non-proliferative can be found in sites where they usually form secondary lesions or in sites where they never do. Thus despite being able to disseminate these DTCs are “growth-suppressed” by certain target organ microenvironments. Several mechanisms are proposed to explain clinical dormancy. The absence of proliferation markers in DTCs in patients and experimental models suggest that solitary DTCs are quiescent. However, angiogenic dormancy or immune system-mediated tumor mass dormancy might also be responsible for maintaining residual disease dormant.
At least three potential scenarios might explain DTC dormancy. 1) DTCs from invasive cancers activate stress signals in response to the dissemination process and/or a growth suppressive target organ microenvironment, inducing dormancy. 2) Therapy and/or microenvironmental stress conditions acting on PT cells carrying specific gene signatures primes newly DTCs to enter dormancy. Thus, specific PT “stress microenvironments” might trigger long-term DTC dormancy. 3) Lesions pathologically defined as non-invasive carry cells able to undergo micro-invasion and disseminate. Here although these DTCs were able to disseminate and survive they are unfit for expansion in secondary sites. Perhaps occasional cell divisions allow them to progress via epigenetic and genetic pathways to a fully metastatic cell able to grow in secondary sites. We propose that DTC dormancy is ultimately a survival strategy that when blocked will prevent metastasis. Below we expand on the first two scenarios. For additional details on the third scenario please see. DTC dormancy and the target organ microenvironment. Solitary DTCs in target organs establish interactions with the ECM, immune cells and the vascular system. This and the pattern of metastasis proposed by the seed and soil theory suggests that microenvironments in the target organ can determine metastatic growth vs. dormancy. Our work shows that in squamous carcinoma cells (HEp3) reduced urokinase (uPA) receptor (uPAR) expression deactivated α5β1-integrins and this made these cells incapable of binding efficiently to fibronectin. This resulted in reduced Src-FAK and EGFR signaling but also in p38α/β activation. This caused these tumor cells to enter a state of dormancy characterized by a prolonged G0-G1 arrest. Others have reproduced these findings showing that loss of β1-integrin or FAK signaling in breast cancer models can also induce dormancy and that Src-MLKC signaling can prevent dormancy. In addition, an enriched collagen-I lung microenvironment can trigger intravenously delivered tumor cells to exit from dormancy as solitary cells. On the other hand environments rich in fibrillar collagen-I can induce quiescence of melanoma cells via activation of the discoidin domain receptor 2 and p15INK4b induction. These results imply that stress signaling induced either by therapies or by a restrictive (i.e. fibrotic or non-fibrotic target tissues depending on the tumor type) tissue microenvironments could activate dormancy (or its interruption) in DTCs.
Our work in the HEp3 model also revealed that activation of p38α/β, while inhibiting ERK1/2 signaling activates an unfolded protein response (UPR). These signals induce survival and quiescence of dormant HEp3 (D-HEp3) cells. D-HEp3 cells in vivo enter a deep G0-G1 arrest due to p21 and p27 upregulation. At least 3 transcription factors (TFs), p53, BHLHB3/41/Sharp1 and NR2F1 were induced by p38α/β and required for dormancy in vivo. Bone marrow derived dormant HEp3 cells displayed a low ERK/p38 signaling ratio and induction of these TFs. Interestingly, metastasis suppressor genes (MSGs) like MKK4 and MKK6 activate p38, BHLHB3 is a target of p38 and Nm23-H1 (another MSG) appears to function via the downregulation of EDG2 LPA receptor signaling through ERK1/2. Thus, different mechanisms converge on the regulation of the ERK/p38 signaling ratio to dictate DTC fate. Overall these studies provide clues as to potential mechanisms to induce or maintain dormancy or eradicate DTCs by targeting their survival signals.
Primary tumor microenvironments as determinants of DTC fate. Gene signatures identified in PTs predict long-term metastatic relapse more than a decade later and in the absence of the PT carrying the signature. Gene profiles from the tumor stroma also predict patient outcome. This suggests that a crosstalk between PT cells and their microenvironment in primary sites can dictate distant disease progression. One interpretation is that genes signatures in the PT and the microenvironment determine DTC fate. Since symptomatic metastasis show homogeneous progression, the gene signatures in the PT might provide information on how those individual or groups of genes influence dormancy of DTCs. Modeling how the genes influence DTC survival and quiescence or subsequent angiogenesis or interaction with the immune system might reveal how they regulate minimal residual disease. Importantly, determining whether signatures derived from circulating tumor cells (CTCs) (i.e. recently intravasated tumor cells) are more or equally informative than the PT signatures might justify characterizing CTCs vs. DTCs (i.e. CTCs that already lodged and reside in target organs).
Another potential example of the primary tumor influencing DTC behavior proposed that CTCs might return to the PT in a self-seeding process and this helps “breed” more aggressive variants that colonize target organs. These studies showed that aggressive variants of MDA-MB-231 breast cancer cells were highly efficient in disseminating and cross-seeding contra lateral tumors. The less aggressive variants of different cancer cell lines were less efficient in the seeding self/cross-seeding process. These data suggest that development of a more aggressive metastatic progeny requires the ability of PTs to attract their own CTCs back and that these tumor cells can efficiently re-colonize the PT. It is unclear how this happens after PT surgery, but it is possible that these events take place when multiple metastases co-exist. It will be interesting to determine whether CTCs from patients with early and advanced lesions carry already these “self-seeding” signatures.
An additional scenario of a PT microenvironment influencing DTC behavior is that of systemic tumor instigation. This model proposes that PTs can influence the growth of otherwise-indolent DTCs or micrometastases. This is though to occur by mobilizing bone-marrow cells into the stroma of the indolent lesions. These results suggest that growth and proliferation of poorly aggressive tumors (dormant DTCs and/or micrometastasisβ) can be regulated on a systemic level by endocrine factors released by certain instigating tumors. However, it remains unclear how metastasis are instigated years or decades after patients underwent PT surgery.
Regardless of their origin we propose that characterization of DTCs would be the most relevant because they carry the aggregate information of their origin, how treatment influenced their adaptation and/or selection and ultimately how the target organ dictated their adaptation and/or selection. The challenges presented by the problem of cancer dormancy are significant and studying DTCs and dormant disease is difficult. But the benefits of unraveling the inherent complexity of this step of metastasis biology should be of great impact for cancer patients.
Grant Support: Samuel Waxman Cancer Research Foundation Tumor Dormancy Program, NIH/National Cancer Institute (CA109182, CA163131), NIEHS (ES017146) and NYSTEM grant to J.A.A-G. M.S.S. is supported by a DoD-BCRP Grant-10904826.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY17-02. doi:1538-7445.AM2012-SY17-02