Scientists at the Icahn School of Medicine at Mount Sinai have identified a protein marker that can signpost whether a patient with breast cancer will likely relapse and develop deadly bone metastases, or whether they will survive with a lower risk of developing metastatic disease. Building on previous work in mouse models and in human prostate cancer patients, results from the team’s latest research involving 86 breast cancer patients showed that those who had low levels of the protein marker NR2F1 in breast cancer cells that had spread to their bone marrow always developed metastatic bone cancer, and had poor survival. In contrast, patients who had high levels of NR2F1 (NR2F1high) in these disseminated tumor cells (DTCs) in their bone marrow were less likely to develop metastases, and they survived much longer. The findings also indicated that decreasing levels of NR2F1 in DTCs from sequential bone marrow isolates signalled that the patients would soon relapse.
“High levels of NR2F1 in bone marrow DTCs is associated with a clear patient survival advantage,” comments research lead Julio Aguirre-Ghiso, Ph.D., speaking to ClinicalOMICS. “Our study found that about 57% of patients with high NR2F1 levels [NR2F1high] in these cells went on to develop relapse and 29% bone metastasis, whereas 100% of patients who were NR2F1low developed systemic relapse or died of breast cancer and 67% had bone metastasis. The question is, whether we can use this marker to reliably predict which patients with DTCs in the bone marrow will need aggressive therapy to counter the development of metastatic disease, and for which patients chemotherapy may be unnecessary because the cancer cells in their bone marrow will remain dormant, and we can instead monitor them for any warning changes to NR2F1 levels and/or offer other therapies.”
Aguirre-Ghiso is director of Solid Tumor and Metastasis Research, co-leader of the Cancer Mechanisms Program, director of Head and Neck Cancer Basic Research, and professor of Oncological Sciences, Otolaryngology, and Medicine (Hematology and Medical Oncology) at The Tisch Cancer Institute at the Icahn School of Medicine.” His team, together with Oslo University Hospital researchers headed by Bjørn Naume, M.D., just published their findings in Breast Cancer Research, in a paper entitled, “NR2F1 stratifies dormant disseminated tumor cells in breast cancer patients.”
Some breast cancer patients relapse quite soon after surgery and first-line chemotherapy, while others might remain disease-free for many years, and some may not relapse at all, the authors explain. This suggests that even small numbers of cancer cells that have spread away from the initial tumor site and escape surgery and chemotherapy can remain in a dormant state for many years, but still retain the plasticity to reactivate and develop into metastatic tumors at a later stage.
Prior studies in breast cancer patients had found that while the presence of DTCs in bone marrow is a strong indicator that the disease will recur within 5 years, more than half of these DTC-positive patients remain relapse free. In an ideal world clinicians would have reliable prognostic indicators to help identify those patients with bone marrow DTCs who are likely to develop metastases, and those whose disease wont progress.
Work by the Tisch Cancer Institute had been focused on NR2F1 as a potential prognostic indicator of cancer metastasis for a number of years. The protein is an orphan nuclear receptor of the retinoic acid family, which prior work by Dr. Aguirre-Ghiso had shown is commonly present at low levels in human primary and metastatic cancers, and which in animal models is upregulated in DTCs that are dormant. Study data indicate that NR2F1 triggers cancer cells to enter this dormant, or quiescent state.
“NR2F1 is an important lineage commitment regulator in development,” Aguirre-Ghiso explained to ClinicalOMICS. “It’s the perfect candidate for a regulator of cancer cell dormancy. The NR2F1 protein essentially tells cells when to stop dividing and enter quiescence, and is controlled by epigenetic mechanisms that switch NR2F1 off when cell growth is needed, and upregulate its expression when cells need to stop dividing and enter dormancy. NR2F1 also regulates some pluripotency genes, which would explain why the dormant cells remain plastic and can be reactivated given the right triggers in the microenvironment.”
The team’s prior research had also shown that when cancer cells lacking in NR2F1 are treated using a combination of the demethylating agent azacitidine, and retinoic acid, which induces cell differentiation – both drugs are used in the clinic – NR2F1 expression is switched on and the cells enter dormancy. “This finding led to the start of a clinical trial that is now ongoing at Mount Sinai, which is combining azacitidine and retinoic acid as a treatment for high risk prostate cancer patients, to see if we can extend their lifespan by inducing or sustaining the cancer’s dormant state.”
The latest reported research in breast cancer was designed to investigate whether NR2F1 can be used to profile patients and determine who may or may not benefit from treatment, Dr. Aguirre-Ghiso continued. “Bjorn had been monitoring DTCs in the bone marrow of breast cancer patients in three Norwegian cohorts to try and correlate the presence of these cells with the success of chemotherapy protocols. What he found and published, was that patients cleared for bone marrow DTCs after therapy survived longer, while a group of patients with bone marrow DTCs would relapse at some point after initial therapy, whether earlier or later. What he didn’t have was any way of telling which patients with DTCs would relapse and so would benefit from chemotherapy to prevent metastasis, and which patients could be spared therapy or offered other treatments because their DTCs would remain dormant. In the joint work just published we adapted the dormancy marker protocol my team had used in the mouse and human squamous carcinoma or prostate cancer patient samples, but in this case we looked at NR2F1 levels in breast cancer DTCs from bone marrow aspirates, at single cell resolution, and correlated expression of the protein with relapse and outcome.”
Aguirre-Ghiso suggests the findings that high NR2F1 levels correlate with continued cell dormancy strongly suggest that profiling for NR2F1 in bone marrow DTCs could help to direct treatment in breast cancer patients. “The data shows the potential feasibility of using DTC NR2F1 levels in the patient to eventually make decisions on whether to continue treatment, or whether to monitor and spare patients from unnecessary, highly toxic chemotherapy or use in the future new therapies that maintain dormancy or kill dormant cells.” And by taking bone marrow samples from NR2F1high patients over time, clinicians could monitor levels of the protein in DTCs, and if they started to drop this would indicate that the cells are coming out of dormancy. Aggressive anticancer therapy could be started to prevent metastasis developing. “Our results should be interpreted with caution due to the small number of patients, but it is a first step that warrants additional studies to increase the number of patients and make the correlation even more robust.”
The team also hopes that the published research will provide a solid foundation for oncologists to consider breast cancer trials in which drugs such as azacitidine and retinoic acid are administered to patients with NR2F1low DTCs to switch cells into a dormant state, and so prolong time to relapse, outcome and lifespan. Similarly, patients with NR2F1high DTCs may also benefit from such therapy as a dormancy maintenance therapy “Breast cancer clinical practice is already served by excellent standards of care, and there are very effective drugs, such as anti-estrogens, which significantly improve breast cancer survival. Monitoring NR2F1 in bone marrow DTCs could become an alternative, parallel approach to keeping the cancer cells dormant.”
The ongoing prostate cancer study with azacitidine and retinoic acid, meanwhile, could generate interpretable data in another year or so, Dr. Aguirre-Ghiso continues. “The trial is profiling NR2F1 levels in circulating tumor cells to see if they respond to therapy with the two drugs. We are looking at NR2F1 levels both before and after therapy, and measuring other factors, such as TGFbeta2, BMPs and cytokines that may indicate the induction of cell dormancy, so we can see if monitoring these biomarkers tells us anything about the systemic response to treatment.”
Prostate cancer, like some forms of breast cancer, also commonly metastasizes to the bone, and at the end of the trial patients will be given the option of a bone marrow aspirate so that the presence of DTCs and levels of biomarkers, including NR2F1, can be evaluated for prognostic insight. “Prostate cancer clinicians have embraced the trial,” Dr. Aguirre-Ghiso stated. “Although there are new drugs for treating this form of cancer, a certain group of patients will inevitably relapse with bone metastases at some point, and oncologists have been very eager to engage in a study that is focused on helping to slow disease in that population.”
What triggers DTCs to come out of dormancy remains another question to be answered, he acknowledged. “Relapse in patients is really stochastic. We and other researchers are exploring the possibility that changes in the bone marrow due to inflammation or aging, for example, may prompt relapse. It’s also possible that with aging, the clonal expansion of certain lineages, or the development of bone marrow fibrosis, may alter the microenvironment, which sends signals that reactivate DTCs so that they escape from dormancy and start proliferating. This is something that we are exploring at the moment.”
