Bone marrow NG2+/Nestin+ mesenchymal stem cells drive DTC dormancy via TGF-β2

Abstract

In the bone marrow (BM) microenvironment, NG2 + /Nestin + mesenchymal stem cells (MSCs) promote hematopoietic stem cell (HSC) quiescence 1,2 . Importantly, the BM can also harbour disseminated tumour cells (DTCs) from multiple cancers, which, like HSCs, can remain dormant 3 . The BM signals are so growth-restrictive that dormant BM DTCs can persist for years to decades only to awaken and fuel lethal metastasis 3-10 . The mechanisms and niche components regulating DTC dormancy remain largely unknown. Here, we reveal that periarteriolar BM-resident NG2 + /Nestin + MSCs can instruct breast cancer (BC) DTCs to enter dormancy. NG2 + /Nestin + MSCs produce TGFβ2 and BMP7 and activate a quiescence pathway dependent on TGFBRIII and BMPRII, which via p38-kinase result in p27-CDK inhibitor induction. Importantly, genetic depletion of the NG2 + /Nestin + MSCs or conditional knock-out of TGFβ2 in the NG2 + /Nestin + MSCs led to awakening and bone metastatic expansion of otherwise dormant p27 + /Ki67 -DTCs. Our results provide a direct proof that HSC dormancy niches control BC DTC dormancy. Given that aged NG2 + /Nestin + MSCs can lose homeostatic control of HSC dormancy, our results suggest that aging or extrinsic factors that affect the NG2 + /Nestin + MSC niche may result in a break from dormancy and BC bone relapse.Metastases, which are derived from disseminated tumour cells (DTCs), are the major source of cancer-related deaths from solid cancers 11 . Ample evidence supports that post-extravasation DTCs can remain in a dormant state from prolonged periods dictating the timing of metastasis initiation 3 .Since years to decades can lapse before dormant DTCs emerge as overt lesions, we postulate that targeting their biology is the shortest path to change patient outcomes by curtailing DTC conversion into metastasis. However, to achieve this goal we must understand the cancer cell intrinsic and microenvironmental mechanisms that control DTC dormancy and reactivation.The bone marrow (BM) is a common site where dormant DTCs are found and where metastasis can develop in various cancers after prolonged periods of clinical "remission" [3][4][5][6][7][8][9][10] . In trying to understand how the BM microenvironment might control DTC dormancy, we 9,12 and others 3,5,13,14 found that in both humans and mice, this microenvironment is a highly restrictive site for metastasis initiation. This is due to the presence of several cues, such as TGFβ2 12 , BMP7 15,16 , and LIF 14,20 , which induce DTC dormancy in different cancer types. Studies in mostly 2D or 3D in vitro models, proposed that mesenchymal stem cells (MSCs) 21 , vascular endothelial cells 13,22 and/or osteoblasts 18,23,24 may be the source of dormancy cues for different cancers. However, the function of such niche cells in vivo has not been formally tested.There is a long-standing hypothesis that the niches that control hematopoietic stem cell (HSC) dormancy may instruct DTCs to become dormant 25 . A prior study in prostate cancer has drawn a

Epigenetic reprogramming of DCCs into dormancy suppresses metastasis via restored TGFβ–SMAD4 signaling

“…However, we did not observe any considerable change in the expression or phosphorylation of the SMAD1/5 pathway as reported for TGFβ2 24 (Figure 3a) , arguing that the AZA+atRA reprogramming may induce a canonical pathway activation. We also documented an increase in the expression of TGFβRI and TGFβRIII (Figure S4b) , both previously implicated in DCC dormancy in the BM and lung 23, 24 . Upon AZA+atRA reprogramming, we also detected a strong increase in the phosphor-p38 and p27, while P-Erk1/2 or total Erk1/2 remained constant, leading to a low ERK/p38 activity ratio 14, 32 (Figure 3a) .…”

“…We observed that all three SMADs are, as expected, low in malignant T-HEp3 cells, and AZA+atRA treatment caused a significant increase in the total expression level of SMAD2, SMAD3, and SMAD4 (Figure 3a) . We had published that TGFβ2 induced DCC dormancy in the bone marrow via an activated P-p38 as well as SMAD2 and SMAD1/5 pathway, which induced expression of p27 leading to quiescence 23, 24 . Upon AZA+atRA reprogramming, we observed an increase in the mRNA levels of TGFβ2 14 , along with TGFβ1 and TGFβ3 ( Figure S4a) .…”

“…In addition to these pathways, TGF receptor signaling is one of the significantly enriched pathways in the Wikipathways database (Figure S1d). Many studies, including our previous reports, have shown that TGF/SMAD pathway plays an important role in normal stem cell and cancer cell dormancy, where a higher ratio of TGF2/TGF1 promotes dormancy via specific TGF receptors 21,22,23,24 . Further, we performed ChIP Enrichment Analysis 25 using the differentially expressed genes upon AZA+atRA reprogramming, and found a significant enrichment of SMAD2 and SMAD3, accounting for 21.7% (51/235) of differentially expressed up-regulated genes (Figure 1c).…”

Prostate Cancer Dormancy and Reactivation in Bone Marrow

“…Cancer-induced bone changes seem to take place when DCCs reactivate and during the proliferation period. Specifically, reactivated DCCs can lead to the differentiation of osteoclastic precursors and fuel the process of osteolytic metastasis [ 33 , 34 , 35 ]. In contrast, the interplay between dormant cells and cellular components of bone appears to favor osteoclasts and osteoblasts working in a balanced manner, similar to that of a healthy individual.…”

“…Periarteriolar BM-resident NG2+/nestin+ MSCs guide BCa DCCs to enter dormancy by producing TGFβ2 and bone morphogenetic protein (BMP7), which activates a quiescence pathway dependent on TGFFBRIII and BMPRII. Depletion of the NG2+/nestin+ MSCs or knockout (KO) of TGFβ2 specifically from NG2+/nestin+ MSCs leads to metastatic outgrowth in BM [ 33 ].…”