Corrigendum: Mechanism of early dissemination and metastasis in Her2+ mammary cancer.

This corrects the article DOI: 10.1038/nature20609.

Mechanism of early dissemination and metastasis in Her2+ mammary cancer.

Metastasis is the leading cause of cancer-related deaths; metastatic lesions develop from disseminated cancer cells (DCCs) that can remain dormant. Metastasis-initiating cells are thought to originate from a subpopulation present in progressed, invasive tumours. However, DCCs detected in patients before the manifestation of breast-cancer metastasis contain fewer genetic abnormalities than primary tumours or than DCCs from patients with metastases. These findings, and those in pancreatic cancer and melanoma models, indicate that dissemination might occur during the early stages of tumour evolution. However, the mechanisms that might allow early disseminated cancer cells (eDCCs) to complete all steps of metastasis are unknown. Here we show that, in early lesions in mice and before any apparent primary tumour masses are detected, there is a sub-population of Her2+p-p38lop-Atf2loTwist1hiE-cadlo early cancer cells that is invasive and can spread to target organs. Intra-vital imaging and organoid studies of early lesions showed that Her2+ eDCC precursors invaded locally, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent epithelial-mesenchymal transition (EMT)-like dissemination program but without complete loss of the epithelial phenotype, which was reversed by Her2 or Wnt inhibition. Notably, although the majority of eDCCs were Twist1hiE-cadlo and dormant, they eventually initiated metastasis. Our work identifies a mechanism for early dissemination in which Her2 aberrantly activates a program similar to mammary ductal branching that generates eDCCs that are capable of forming metastasis after a dormancy phase.

Early dissemination seeds metastasis in breast cancer.

Accumulating data suggest that metastatic dissemination often occurs early during tumour formation, but the mechanisms of early metastatic spread have not yet been addressed. Here, by studying metastasis in a HER2-driven mouse breast cancer model, we show that progesterone-induced signalling triggers migration of cancer cells from early lesions shortly after HER2 activation, but promotes proliferation in advanced primary tumour cells. The switch from migration to proliferation was regulated by increased HER2 expression and tumour-cell density involving microRNA-mediated progesterone receptor downregulation, and was reversible. Cells from early, low-density lesions displayed more stemness features, migrated more and founded more metastases than cells from dense, advanced tumours. Notably, we found that at least 80% of metastases were derived from early disseminated cancer cells. Karyotypic and phenotypic analysis of human disseminated cancer cells and primary tumours corroborated the relevance of these findings for human metastatic dissemination.

NR2F1 stratifies dormant disseminated tumor cells in breast cancer patients.

BACKGROUND: The presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor in early breast cancer but does not uniformly predict outcome. Tumor cells can persist in a quiescent state over time, but clinical studies of markers predicting the awakening potential of DTCs are lacking. Recently, experiments have shown that NR2F1 (COUP-TF1) plays a key role in dormancy signaling. METHODS: We analyzed the NR2F1 expression in DTCs by double immunofluorescence (DIF) staining of extra cytospins prepared from 114 BM samples from 86 selected DTC-positive breast cancer patients. Samples collected at two or more time points were available for 24 patients. Fifteen samples were also analyzed for the proliferation marker Ki67. RESULTS: Of the patients with detectable DTCs by DIF, 27% had ≥ 50% NR2F1high DTCs, chosen a priori as the cut-off for "dormant profile" classification. All patients with systemic relapse within 12 months after BM aspiration carried ≤ 1% NR2F1high DTCs, including patients who transitioned from having NR2F1high-expressing DTCs in previous BM samples. Of the patients with serial samples, half of those with no relapse at follow-up had ≥ 50% NR2F1high DTCs in the last BM aspiration analyzed. Among the 18 relapse-free patients at the time of the last DTC-positive BM aspiration with no subsequent BM analysis performed, distant disease-free intervals were favorable for patients carrying ≥ 50% NR2F1high DTCs compared with those with predominantly NR2F1low DTCs (p = 0.007, log-rank). No survival difference was observed by classification according to Ki67-expressing DTCs (p = 0.520). CONCLUSIONS: Our study translates findings from basic biological analysis of DTC dormancy to the clinical situation and supports further clinical studies of NR2F1 as a marker of dormancy.

Time-lapsed, large-volume, high-resolution intravital imaging for tissue-wide analysis of single cell dynamics.

Pathologists rely on microscopy to diagnose disease states in tissues and organs. They utilize both high-resolution, high-magnification images to interpret the staining and morphology of individual cells, as well as low-magnification overviews to give context and location to these cells. Intravital imaging is a powerful technique for studying cells and tissues in their native, live environment and can yield sub-cellular resolution images similar to those used by pathologists. However, technical limitations prevent the straightforward acquisition of low-magnification images during intravital imaging, and they are hence not typically captured. The serial acquisition, mosaicking, and stitching together of many high-resolution, high-magnification fields of view is a technique that overcomes these limitations in fixed and ex vivo tissues. The technique however, has not to date been widely applied to intravital imaging as movements caused by the living animal induce image distortions that are difficult to compensate for computationally. To address this, we have developed techniques for the stabilization of numerous tissues, including extremely compliant tissues, that have traditionally been extremely difficult to image. We present a novel combination of these stabilization techniques with mosaicked and stitched intravital imaging, resulting in a process we call Large-Volume High-Resolution Intravital Imaging (LVHR-IVI). The techniques we present are validated and make large volume intravital imaging accessible to any lab with a multiphoton microscope.

Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer.

While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gßγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gßγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.

Epigenetic and Pluripotency Aspects of Disseminated Cancer Cells During Minimal Residual Disease.

Our understanding of the minimal residual disease (MRD) in solid cancers indicates that it can persist in the system for years or even decades. We now know that the persistence of MRD might depend on the dormancy of the disseminated cancer cells (DCCs). Once DCCs exit dormancy, they become metastatic and the survival rates of the patients inevitably decrease. Thus, innovative treatments are required to extend the asymptomatic phase of MRD after the initial therapeutic intervention. With the latest advances in cancer research, there is a greater need to explore and understand the biology, timing of dissemination, and origin of DCCs during tumor progression. These important aspects of DCCs impact the selection, design, administration, and timing of effective therapies. Herein, we summarize the current understanding of MRD biology in solid tumors, with a focus on epigenetics and pluripotency, presenting an overall view of the direction the field is taking to reach the goal of reducing cancer-related mortalities that result from metastasis.

How much do we know about the metastatic process?

Cancer cells can leave their primary sites and travel through the circulation to distant sites, where they lodge as disseminated cancer cells (DCCs), even during the early and asymptomatic stages of tumor progression. In experimental models and clinical samples, DCCs can be detected in a non-proliferative state, defined as cellular dormancy. This state can persist for extended periods until DCCs reawaken, usually in response to niche-derived reactivation signals. Therefore, their clinical detection in sites like lymph nodes and bone marrow is linked to poor survival. Current cancer therapy designs are based on the biology of the primary tumor and do not target the biology of the dormant DCC population and thus fail to eradicate the initial or subsequent waves of metastasis. In this brief review, we discuss the current methods for detecting DCCs and highlight new strategies that aim to target DCCs that constitute minimal residual disease to reduce or prevent metastasis formation. Furthermore, we present current evidence on the relevance of DCCs derived from early stages of tumor progression in metastatic disease and describe the animal models available for their study. We also discuss our current understanding of the dissemination mechanisms utilized by genetically less- and more-advanced cancer cells, which include the functional analysis of intermediate or hybrid states of epithelial-mesenchymal transition (EMT). Finally, we raise some intriguing questions regarding the clinical impact of studying the crosstalk between evolutionary waves of DCCs and the initiation of metastatic disease.

Oncogenic enhancers prime quiescent metastatic cells to escape NK immune surveillance by eliciting transcriptional memory.

Metastasis arises from disseminated tumour cells (DTCs) that are characterized by intrinsic phenotypic plasticity and the capability of seeding to secondary organs. DTCs can remain latent for years before giving rise to symptomatic overt metastasis. In this context, DTCs fluctuate between a quiescent and proliferative state in response to systemic and microenvironmental signals including immune-mediated surveillance. Despite its relevance, how intrinsic mechanisms sustain DTCs plasticity has not been addressed. By interrogating the epigenetic state of metastatic cells, we find that tumour progression is coupled with the activation of oncogenic enhancers that are organized in variable interconnected chromatin domains. This spatial chromatin context leads to the activation of a robust transcriptional response upon repeated exposure to retinoic acid (RA). We show that this adaptive mechanism sustains the quiescence of DTCs through the activation of the master regulator SOX9. Finally, we determine that RA-stimulated transcriptional memory increases the fitness of metastatic cells by supporting the escape of quiescent DTCs from NK-mediated immune surveillance. Overall, these findings highlight the contribution of oncogenic enhancers in establishing transcriptional memories as an adaptive mechanism to reinforce cancer dormancy and immune escape, thus amenable for therapeutic intervention.

Regulation of tumor cell dormancy by tissue microenvironments and autophagy.

The development of metastasis is the major cause of death in cancer patients. In certain instances, this occurs shortly after primary tumor detection and treatment, indicating these lesions were already expanding at the moment of diagnosis or initiated exponential growth shortly after. However, in many types of cancer, patients succumb to metastatic disease years and sometimes decades after being treated for a primary tumor. This has led to the notion that in these patients residual disease may remain in a dormant state. Tumor cell dormancy is a poorly understood phase of cancer progression and only recently have its underlying molecular mechanisms started to be revealed. Important questions that remain to be elucidated include not only which mechanisms prevent residual disease from proliferating but also which mechanisms critically maintain the long-term survival of these disseminated residual cells. Herein, we review recent evidence in support of genetic and epigenetic mechanisms driving dormancy. We also explore how therapy may cause the onset of dormancy in the surviving fraction of cells after treatment and how autophagy may be a mechanism that maintains the residual cells that are viable for prolonged periods.

5-Azacytidine- and retinoic-acid-induced reprogramming of DCCs into dormancy suppresses metastasis via restored TGF-ß-SMAD4 signaling.

Disseminated cancer cells (DCCs) in secondary organs can remain dormant for years to decades before reactivating into overt metastasis. Microenvironmental signals leading to cancer cell chromatin remodeling and transcriptional reprogramming appear to control onset and escape from dormancy. Here, we reveal that the therapeutic combination of the DNA methylation inhibitor 5-azacytidine (AZA) and the retinoic acid receptor ligands all-trans retinoic acid (atRA) or AM80, an RARα-specific agonist, promotes stable dormancy in cancer cells. Treatment of head and neck squamous cell carcinoma (HNSCC) or breast cancer cells with AZA+atRA induces a SMAD2/3/4-dependent transcriptional program that restores transforming growth factor ß (TGF-ß)-signaling and anti-proliferative function. Significantly, either combination, AZA+atRA or AZA+AM80, strongly suppresses HNSCC lung metastasis formation by inducing and maintaining solitary DCCs in a SMAD4+/NR2F1+ non-proliferative state. Notably, SMAD4 knockdown is sufficient to drive resistance to AZA+atRA-induced dormancy. We conclude that therapeutic doses of AZA and RAR agonists may induce and/or maintain dormancy and significantly limit metastasis development.

Microenvironments dictating tumor cell dormancy.

The mechanisms driving dormancy of disseminated tumor cells (DTCs) remain largely unknown. Here, we discuss experimental evidence and theoretical frameworks that support three potential scenarios contributing to tumor cell dormancy. The first scenario proposes that DTCs from invasive cancers activate stress signals in response to the dissemination process and/or a growth suppressive target organ microenvironment inducing dormancy. The second scenario asks whether therapy and/or micro-environmental stress conditions (e.g. hypoxia) acting on primary tumor cells carrying specific gene signatures prime new DTCs to enter dormancy in a matching target organ microenvironment that can also control the timing of DTC dormancy. The third and final scenario proposes that early dissemination contributes a population of DTCs that are unfit for immediate expansion and survive mostly in an arrested state well after primary tumor surgery, until genetic and/or epigenetic mechanisms activate their proliferation. We propose that DTC dormancy is ultimately a survival strategy that when targeted will eradicate dormant DTCs preventing metastasis. For these non-mutually exclusive scenarios we review experimental and clinical evidence in their support.

NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

Cancer cells can disseminate during very early and sometimes asymptomatic stages of tumor progression. Though biological barriers to tumorigenesis have been identified and characterized, the mechanisms that limit early dissemination remain largely unknown. We report here that the orphan nuclear receptor nuclear receptor subfamily 2, group F, member 1 (NR2F1)/COUP-TF1 serves as a barrier to early dissemination. NR2F1 expression was decreased in patient ductal carcinoma in situ (DCIS) samples. High-resolution intravital imaging of HER2+ early-stage cancer cells revealed that loss of function of NR2F1 increased in vivo dissemination and was accompanied by decreased E-cadherin expression, activation of wingless-type MMTV integration site family, member 1 (WNT)-dependent ß-catenin signaling, disorganized laminin 5 deposition, and increased expression of epithelial-mesenchymal transition (EMT) genes such as twist basic helix-loop-helix transcription factor 1 (TWIST1), zinc finger E-box binding homeobox 1 (ZEB1), and paired related homeobox 1 (PRRX1). Furthermore, downregulation of NR2F1 promoted a hybrid luminal/basal phenotype. NR2F1 expression was positively regulated by p38α signaling and repressed by HER2 and WNT4 pathways. Finally, early cancer cells with NR2F1LOW/PRRX1HIGH staining were observed in DCIS samples. Together, these findings reveal the existence of an inhibitory mechanism of dissemination regulated by NR2F1 in early-stage breast cancer cells. SIGNIFICANCE: During early stages of breast cancer progression, HER2-mediated suppression of NR2F1 promotes dissemination by inducing EMT and a hybrid luminal/basal-like program.

An NR2F1-specific agonist suppresses metastasis by inducing cancer cell dormancy.

We describe the discovery of an agonist of the nuclear receptor NR2F1 that specifically activates dormancy programs in malignant cells. The agonist led to a self-regulated increase in NR2F1 mRNA and protein and downstream transcription of a novel dormancy program. This program led to growth arrest of an HNSCC PDX line, human cell lines, and patient-derived organoids in 3D cultures and in vivo. This effect was lost when NR2F1 was knocked out by CRISPR-Cas9. RNA sequencing revealed that agonist treatment induces transcriptional changes associated with inhibition of cell cycle progression and mTOR signaling, metastasis suppression, and induction of a neural crest lineage program. In mice, agonist treatment resulted in inhibition of lung HNSCC metastasis, even after cessation of the treatment, where disseminated tumor cells displayed an NR2F1hi/p27hi/Ki-67lo/p-S6lo phenotype and remained in a dormant single-cell state. Our work provides proof of principle supporting the use of NR2F1 agonists to induce dormancy as a therapeutic strategy to prevent metastasis.

Primary tumor associated macrophages activate programs of invasion and dormancy in disseminating tumor cells.

Metastases are initiated by disseminated tumor cells (DTCs) that colonize distant organs. Growing evidence suggests that the microenvironment of the primary tumor primes DTCs for dormant or proliferative fates. However, the manner in which this occurs remains poorly understood. Here, using the Window for High-Resolution Intravital Imaging of the Lung (WHRIL), we study the live lung longitudinally and follow the fate of individual DTCs that spontaneously disseminate from orthotopic breast tumors. We find that spontaneously DTCs have increased levels of retention, increased speed of extravasation, and greater survival after extravasation, compared to experimentally metastasized tumor cells. Detailed analysis reveals that a subset of macrophages within the primary tumor induces a pro-dissemination and pro-dormancy DTC phenotype. Our work provides insight into how specific primary tumor microenvironments prime a subpopulation of cells for expression of proteins associated with dissemination and dormancy.

A novel cross-talk in diacylglycerol signaling: the Rac-GAP beta2-chimaerin is negatively regulated by protein kinase Cdelta-mediated phosphorylation.

Although the family of chimaerin Rac-GAPs has recently gained significant attention for their involvement in development, cancer, and neuritogenesis, little is known about their molecular regulation. Chimaerins are activated by the lipid second messenger diacylglycerol via their C1 domain upon activation of tyrosine kinase receptors, thereby restricting the magnitude of Rac signaling in a receptor-regulated manner. Here we identified a novel regulatory mechanism for beta2-chimaerin via phosphorylation. Epidermal growth factor or the phorbol ester phorbol 12-myristate 13-acetate caused rapid phosphorylation of beta2-chimaerin on Ser(169) located in the SH2-C1 domain linker region via protein kinase Cdelta, which retained beta2-chimaerin in the cytosol and prevented its C1 domain-mediated translocation to membranes. Furthermore, despite the fact that Ser(169) phosphorylation did not alter intrinsic Rac-GAP activity in vitro, a non-phosphorylatable beta2-chimaerin mutant was highly sensitive to translocation, and displayed enhanced association with activated Rac, enhanced Rac-GAP activity, and anti-migratory properties when expressed in cells. Our results not only revealed a novel regulatory mechanism that facilitates Rac activation, but also identified a novel mechanism of cross-talk between diacylglycerol receptors that restricts beta2-chimaerin relocalization and activation.

Biochemical characterization of hyperactive beta2-chimaerin mutants revealed an enhanced exposure of C1 and Rac-GAP domains.

Recent studies established that the Rac-GAP beta2-chimaerin plays important roles in development, neuritogenesis, and cancer progression. A unique feature of beta2-chimaerin is that it can be activated by phorbol esters and the lipid second messenger diacylglycerol (DAG), which bind with high affinity to its C1 domain and promote beta2-chimaerin translocation to membranes, leading to the inactivation of the small G-protein Rac. Crystallographic evidence and cellular studies suggest that beta2-chimaerin remains in an inactive conformation in the cytosol with the C1 domain inaccessible to ligands. We developed a series of beta2-chimaerin point mutants in which intramolecular contacts that occlude the C1 domain have been disrupted. These mutants showed enhanced translocation in response to phorbol 12-myristate 13-acetate (PMA) in cells. Binding assays using [(3)H]phorbol 12,13-dibutyrate ([(3)H]PDBu) revealed that internal contact mutants have a reduced acidic phospholipid requirement for phorbol ester binding. Moreover, disruption of intramolecular contacts enhances binding of beta2-chimaerin to acidic phospholipid vesicles and confers enhanced Rac-GAP activity in vitro. These studies suggest that beta2-chimaerin must undergo a conformational rearrangement in order to expose its lipid binding sites and become activated.

Macrophages orchestrate breast cancer early dissemination and metastasis.

Cancer cell dissemination during very early stages of breast cancer proceeds through poorly understood mechanisms. Here we show, in a mouse model of HER2+ breast cancer, that a previously described sub-population of early-evolved cancer cells requires macrophages for early dissemination. Depletion of macrophages specifically during pre-malignant stages reduces early dissemination and also results in reduced metastatic burden at end stages of cancer progression. Mechanistically, we show that, in pre-malignant lesions, CCL2 produced by cancer cells and myeloid cells attracts CD206+/Tie2+ macrophages and induces Wnt-1 upregulation that in turn downregulates E-cadherin junctions in the HER2+ early cancer cells. We also observe macrophage-containing tumor microenvironments of metastasis structures in the pre-malignant lesions that can operate as portals for intravasation. These data support a causal role for macrophages in early dissemination that affects long-term metastasis development much later in cancer progression. A pilot analysis on human specimens revealed intra-epithelial macrophages and loss of E-cadherin junctions in ductal carcinoma in situ, supporting a potential clinical relevance.

Dormancy programs as emerging antimetastasis therapeutic alternatives.

We recently published that the retinoid-responsive gene NR2F1 (nuclear receptor subfamily 2, group F, member 1) mediates postsurgical dormancy of local residual tumor cells and disseminated tumor cells. Importantly, the combination of azacytidine with retinoids induces dormancy of malignant tumor cells by reinstating the NR2F1-regulated gene program. These findings open the door to the development of strategies that may stop minimal residual disease from becoming life-threatening metastases.