Defining the “Metastasome”: Perspectives from the genome and molecular landscape in colorectal cancer for metastasis evolution and clinical consequences

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Review

Defining the “Metastasome”: Perspectives from the genome and molecular landscape in colorectal cancer for metastasis evolution and clinical consequences ⁎

Heike Allgayera,b, , Jörg H. Leupolda,b, Nitin Patila,b a Department of Experimental Surgery – Cancer Metastasis, Medical Faculty Mannheim, Theodor Kutzer Ufer 1-3, 68135, Mannheim, Ruprecht Karls University of Heidelberg, Germany b Centre for Biomedicine and Medical Technology Mannheim (CBTM), Medical Faculty Mannheim, Ludolf-Krehl-Str. 6, 68135, Mannheim, Ruprecht Karls University of Heidelberg, Germany

A R T I C LE I N FO

A B S T R A C T

Keywords: Metastasis Whole genome Colorectal cancer Sequencing Metastasome

Metastasis still poses the highest challenge for personalized therapy in cancer, partly due to a still incomplete understanding of its molecular evolution. We recently presented the most comprehensive whole-genome study of colorectal metastasis vs. matched primary tumors and suggested novel components of disease progression and metastasis evolution, some of them potentially relevant for targeted therapy. In this review, we try to put these findings into perspective with latest discoveries of colleagues and recent literature, and propose a systematic international team effort to collectively define the “metastasome”, a term we introduce to summarize all genomic, epigenomic, transcriptomic, further –omic, molecular and functional characteristics rendering metastases different from primary tumors. Based on recent discoveries, we propose a revised metastasis model for colorectal cancer which is based on a common ancestor clone, early dissemination but flexible early or late stage clonal separation paralleling stromal interactions. Furthermore, we discuss hypotheses on site-specific metastasis, colorectal cancer progression, metastasis-targeted diagnosis and therapy, and metastasis prevention based on latest metastasome data.

1. Introduction “Healing is a matter of time, but it is sometimes also a matter of opportunity” (Hippokrates of Kos, 460-370BCE, https://brainyquote. com). Unfortunately, although continuous efforts have been made in research, clinical diagnosis and personalized therapy for quite some time, metastasis still by far is the most frequent cause of cancer-related deaths in our modern world. As compared to, e.g., systematic worldwide networking efforts to decipher the human genome, and the genome of the most frequent primary cancer entities (e.g. within The Cancer Genome Atlas (TCGA), [1–4]), research efforts to combat or even prevent metastasis are still rather fragmented, and a still incomplete understanding of metastasis evolution from the scientific perspective is paralleled by clinical strategies which still cannot be specific enough to target the metastatically relevant tumor cell clones. Potential reasons for this are severalfold; one certainly is that up to a few years ago, experimental models which could convincingly reflect and model the natural development of human cancer metastasis, and its

interactions with realistic (micro-) environmental settings, were rare or still lacking. In translational and clinical research, one major drawback from the authors’ perspective has been the fact that very few hypothesis-generating studies have been conducted at high-quality metastasis tissues of cancer patients. Enumerous studies at large series of primary tumor tissues have been performed in the past [2,5,6] which included patients with clinical metastatic stages; however, also in the latter cases most studies have restricted themselves to studying primary tumor tissues of these patients, without, or rarely, looking at paired samples of the metastatic lesions of these patients. Certainly, a huge general problem in this context is that hardly any center in the world on its own have large numbers of metastasized patient cases available in which samples of resected metastasis lesions together with paired primary tumors and corresponding normal background tissues are at hand. However, latest technological developments in advancing attractive models to study metastasis, and high-throughput technologies including, but not limited to, latest genomic, transcriptomic, proteomic, but also bioinformatics and mathematical modeling platforms, as well

⁎ Corresponding author at: Department of Experimental Surgery, Cancer Metastasis and Vice Director, Centre for Biomedicine and Medical Technology Mannheim (CBTM), Medical Faculty Mannheim, University of Heidelberg, Ludolf-Krehl-Str. 13-17, 68167, Mannheim, Germany. E-mail address: [email protected] (H. Allgayer).

https://doi.org/10.1016/j.semcancer.2019.07.018 Received 28 May 2019; Received in revised form 22 July 2019; Accepted 23 July 2019 1044-579X/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

Please cite this article as: Heike Allgayer, Jörg H. Leupold and Nitin Patil, Seminars in Cancer Biology, https://doi.org/10.1016/j.semcancer.2019.07.018

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sequencing [25] and one using targeted sequencing of cancer-associated genes [26] found a high concordance rate in single mutations between matched primary tumors and metastases; in addition, the former study applied a mathematical model to translate their findings into distance of time, and estimated a much shorter time of development up to clinical diagnosis for the metastasis as compared to the primary [25]. Thus, both of these studies concluded that their data rather supported the linear progression model. In our recent whole genome study, we found that an average of 65% of single nucleotide variants (SNVs)/single mutations are shared between the primary tumor and the corresponding metastasis, the highest percentage in one case being 92%, which could be taken as a support for this model as well. Also, our observation of a higher total number of single mutations in the metastases, a higher percentage of metastasis-specific mutations (19% in average, range 3–42%) and a higher mutational rate in the metastases as compared to the corresponding primaries after truncal separation would support the view of Naxerova et al. [10] that the linear progression model needs to assume an accelerated growth rate of metastases (possibly induced by a faster accumulation of mutations). However, as outlined in the same review, in imaging studies the doubling times of macroscopically visible metastatic lesions are not observed to be too different to the ones of primary tumors, and therefore, the netto growth rate of metastases might be divided in a quite accelerated growth rate in the dissemination- or micrometastatic stage before macroscopic detection, and a slower growth rate in the macroscopic detection stage [10]. Still, however, although the majority of the cases studied in our recent paper might support the view of a rather late-stage truncal separation of the metastasis from the primary, we also had some cases in which the percentage of shared single mutations between both was rather low (down to 32% common SNVs between primary tumor and metastasis), the highest percentage of metastasisspecific SNVs in one case being 42%. In addition, in one case of our study we observed a tumor protein 53 (TP53,also known as p53) mutation in the metastasis only, whereas the corresponding primary tumor showed a large deletion spanning the TP53 regulator ATM serine/ threonine kinase (ATM) [8]. This case could suggest a split already at a late adenoma stage and would, together with the other cases of a low percentage of shared mutations, clearly argue for the parallel progression model. This is supported by observations reported earlier that even non-carcinoma and non-invasive cells can enter blood vessels and the systemic circulation, theoretically being a potential source for a later metastatic lesion [22]. Certainly, given the large level of heterogeneity within tumor lesions we need to view this single case we reported above with the caution that the TP53-mutated clone giving rise to the metastasis might have been present within the corresponding primary tumor, but not contained within the sample we had received from the latter. Still, especially given such a case together with others with evidence for a rather early clonal separation, we would definitely support the possibility of a parallel progression model for colorectal cancer as well, especially since it is known that the dissemination of single tumor cells, and also their survival during dissemination, does not depend on a high number of acquired particular genomic changes or mutations [22]. Moreover, it has been known since even decades that in a majority of the most frequent solid carcinoma entities in humans, early disseminated tumor cells can be detected even in very early primary tumor stages in the blood or bone marrow of these patients, including colorectal cancer [27–33]. Since the 1990s, we already know that particular molecules detected on some of these DTCs such as urokinase-type plasminogen activator receptor (u-PAR) in gastrointestinal cancers, human epidermal growth factor receptor 2 (HER2 or HER2/ neu or c-erbB-2) in breast cancers, etc., correlate with later clinical outcome, specifically tumor recurrence and metastasis [8,34–37]. Therefore, it is very clear that among DTCs, there are particular phenotypes that either already carry the potential for metastasis as being derived as a clone from the primary tumor, or have the capacity to acquire this potential during dissemination, maybe during systemic

as impressive advances in methods such as single-cell sequencing of disseminated tumor cells (DTCs) [7] are highly encouraging and suggest that we might have reached the right point in time when fundamental advances might be possible, seeking to take the right opportunities. With our present review, we would like to encourage and invite scientific and clinical colleagues around the world to initiate concerted efforts to define the “metastasome”. Recent publications including our latest ones [8–14], clearly suggest that metastases, and metastatically capable cancer cell clones, exhibit specific characteristics at the genome level, some of which already are, and in the future might increasingly be, paralleled at the transcriptome, protein, epigenetic and functional level. Putting together collective knowledge of specific molecular characteristics of metastases as the “metastasome” clearly bears the chance that treating, or even preventing, metastasis might become possible. In the following, we describe recent developments regarding the metastasome of colorectal cancer, including our own, and try to put them into context with existing hypotheses on cancer progression and metastasis evolution. While focusing at recent genome findings, we of course would like to be kept in mind that transcriptional, translational and further –omics data, as well as functional and mechanistic knowledge, will need to be added continuously to gradually lead to a collective picture of the “metastasome”. In the final chapters, we try to conclude potential diagnostic or clinical implications from the current “metastasome” perspective. 2. Hypotheses on metastasis, clonal differentiation and dissemination: how are they supported by current metastasome data in colorectal cancer? Different models of the phylogenesis of metastasis have been put forward in the past, ranging from the assumption of a common clone of origin for both the primary tumor and the metastases up to hypotheses of a completely independent genesis of metastasis and a primary tumor. The latter hypothesis had been supported by occasional observations of a “cancer of unknown primary (CUP)” [15] in rather rare cases of cancer patients whose first clinical presentation is a tumor with clear evidence of not having risen from the organ site of its detection (fulfilling the definition of a metastasis), to which a matching primary tumor within another organ is never found. However, especially given the advancement of powerful –omics techniques during the very recent years, it became increasingly possible to match metastatic lesions at least to the tissue of origin in such patients [15,16]. In our recent whole-genome data at resected metastases (all of them in the liver with one exception) and corresponding primary colorectal cancer lesions [8], we found clear evidence for a common ancestor clone of the primary tumor and the corresponding metastasis in all of the 12 cases investigated, without any exception, making hypotheses on a completely independent phylogenesis of a primary tumor and a metastasis highly unlikely, at least for this tumor entity. Still, within recently put forward hypotheses on metastasis evolution which assumed a common ancestor, different models have been suggested, the two main ones being the linear versus the parallel progression model, sometimes also mentioned as a late dissemination versus an early dissemination model [10,17–22]. The parallel progression or early dissemination model, put forward especially by C. Klein in 2009 [22] and supported for breast cancer by very recent evidence of this group in a mouse model [23], hypothesizes that the dissemination of metastatically capable cells by the primary tumor occurs in very early stages of primary tumor development, and that the primary and a secondary lesion therefore are evolving separately thereafter. The linear progression model, as the more “classical” one, assumes metastasis as a sequential event following primary tumor development in one timeline, which is mono-directional [10,24]. Some of the few previous studies comparing colorectal cancer metastases and primary tumors, one using early index-lesion 2

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hybridization (CGH) methods. A report of Kim et al [12] which investigated multiple regions of primary colorectal tumors and liver metastases from five patients, already concluded that not all metastatic colorectal cases support the linear model of tumor progression, but rather parallel progression [46]. Also, looking at the evolution of mutational patterns within particular mutational signatures [47], we reconfirmed the hypothesis of a common ancestor clone with then private mutagenic processes ongoing in both the primary tumor and the metastasis after truncal separation [8]. Specifically, for example, the adenylyl cyclase 1 (AC1) mutational pattern, reflecting a clock-like signature which is especially driven by spontaneous deamination, appeared more truncal and common to both, however, mutations ongoing within the AC3 mutational pattern of DSB-repair (double-strand break repair) and a pattern that share molecular features of breast cancer type susceptibility protein (BRCA)-mutant tumours (BRCAness) were more private to both primary tumor and metastasis, thus rather ongoing after truncal separation. Interestingly, a signature of somatic mutations with a strong connection to BRCAness was described in a subset of breast and pancreatic cancer samples, giving the opportunity to transfer this hypothesis as valid for other cancer entities [47]. Taken together, based on the literature and our recent data, for colorectal cancer we suggest a common ancestor clone between metastasis and primary tumor, early heterogeneous dissemination which is independent from early or late clonal separation, the latter depending on the timing of individual genetic events which are critical for forming a metastasis in interaction with systemic and niche components, and parallel progression after truncal separation between the primary and the metastatic lesion (Fig. 1).

priming in the dissemination process or in the interaction with specific metastatic niches and microenvironments which they encounter on their travel. Since the field of tumor microenvironments was pioneered by the group of Isaac P. Witz and others [38,39], evidence has been increasing continuously that diverse microenvironments, metastatic niches, and, in general, stromal components also undergo a dynamic evolution during the metastasis process, and it is quite likely that the evolution of metastatically capable cells occurs during the stage of dissemination in intense interaction with a parallel evolution of the stroma [40]. In this context, a third suggested model for metastasis of “tumor self-seeding” [10,11,41,42], whereby cancer cells are also being spread by the metastasis and return to the primary tumor to contribute to the dynamics of the genesis/progression of the primary tumor, cannot be ruled out from our present genome data or might even seem quite likely [8], especially in the case of a putative separation at the adenoma stage as described above. This model of self-seeding not necessarily has to be mutually exclusive to linear or parallel progression models, but can be considered to happen in parallel [10,21,22]. How can we reconcile these models for metastasis of colorectal cancer? We think an important issue is to clearly separate the term “dissemination” from “clonal separation”, since some previous papers or reviews might not have done so. Some previous papers also use the synonym of “early” and “late dissemination” for the parallel versus the linear progression model, respectively [22]. Given the abundant evidence from several already early papers before the era of single-cell sequencing, but of course also actual ones [22,35,43], our current suggestion for a model of metastasis at least in colorectal cancer would be an early dissemination of already heterogeneous cell clones, and a clonal separation of the metastatically capable clones from others happening during dissemination (Fig. 1). Clonal separation during dissemination will most likely be, at least in part, phylogenetically pushed by a diversity of systemic priming events (e.g., via vascular endothelial growth factor (VEGF) [44]) and/or the interaction of disseminated cells with parallel developing stromal microenvironments, metastatic niches, perivascular niches, specific organ microenvironments, etc. [40,44], whereby of course the priming of metastatic niches by both disseminated and, over the systemic distance, primary cancer cells (e.g. via exosomes) is possible and likely [45]. The timing of the separation of the metastatically relevant clone(s) from the common ancestor within the whole metastatic process might depend on the (coincidental?) timing of particular genetic/epigenetic changes within this process as to their relevance for metastatic capabilities, which would then rather reflect a “parallel progression” model in cases of earlier clonal separation, or a rather “linear progression” hypothesis in cases of later clonal separation (Fig. 1). In any case, however, from our recent genomic data in the 12 colorectal paired metastasis/primary tumor cases we analysed, we have clear evidence that the metastasis always harbours genomic changes/mutations which are private to the metastasis and not shared with the corresponding primary tumor, and vice versa, so that a parallel development (or “parallel progression”) after truncal separation, regardless of having happened early or late, is true in any instance. The fact of metastasisspecific mutations or genomic lesions which occurred after clonal separation is supported by a single-cell sequencing report of Leung et al. in two colorectal cancer patient primary tumors with paired liver metastases [13], in which these colleagues also found mutations private to the metastases, although these colleagues exclusively want to conclude on a late dissemination model based on their two patients, which we do not share due to the aforementioned reasons. Also, a very recent paper by Vermaat et al having performed targeted exome sequencing, confirms our observations of specific genetic changes being different in hepatic metastases of colorectal cancer patients from their primaries [14]. Other reports [6] might not have detected a high extent of specific changes in metastases as compared to primaries in detail due to method limitations, for example targeted sequencing which was restricted to a particular panel of coding genes, or less sensitive comparative genomic

3. The cancer stem cell hypothesis: hypotheses for the colorectal cancer stem cell based on current metastasome data Since it became obvious that the capacity to generate new tumours can be traced back to a small number of cells within a solid tumour, the cancer stem cell (CSC) hypothesis has been postulated to define the characteristic of this specific subset of cells [48,49]. As a result, numerous studies were carried out and described the presence of CSCs in various solid tumours, like breast, lung, or colorectal cancer [50–53]. Further investigations of their potential contribution to the processes of metastasis resulted in the introduction of the term “metastatic stem cell (MetSC)” [54]. It is speculated that these MetSCs most likely already exist in the primary tumour where they might simply develop from the original CSCs, or that they derive from DTCs that regain the ability to initiate metastatic lesions at a distant site, this reiterating different models on metastasis evolution as discussed above (Fig. 2). However, one of the most essential questions which have not been completely solved yet is whether the CSC is equal to “the metastatically relevant cell”, what characteristics a specific MetSC would need to have and how these would be similar to, or different from, what we assume to be CSCs to date. In this context it is important to recapitulate that metastasis is a highly selective, sequential process with several inter-connected steps and can fail at multiple points within the cascade. In the end, the process always starts with malignant cells that evade from the primary tissue, enter the bloodstream and are capable to settle in a distant tissue to give rise to metastasis [55]. Nevertheless, not every disseminated tumour cell (DTC) becomes automatically a “metastasis initiating cell”, leading to the question if there are specific genetic alterations connected to the metastatic subpopulation of DTCs, and if this signature can be traced back to the primary source of the metastatic clone. Supporting the idea that the metastatically relevant cell already exists in the primary tumour, Stange et al. found a high degree of genetic similarity, with most gains, amplifications and losses of genes being already present in the primary tumours compared to the corresponding metastases [56]. Interestingly, closer investigations revealed that a specific genetic aberration was even connected with an increased 3

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Fig. 1. Revised model of metastasis evolution for colorectal cancer: Based on current data, we propose a common ancestor clone and early dissemination but flexible early or late stage clonal separation, reconciling earlier hypotheses of “parallel” or “linear progression” [10,22]. The timing of clonal separation to generate a metastatically capable clone depends on the sequence of genomic and further events private to the respective cancer cell clone, and stromal interactions which parallel the dissemination of cancer cells. These include systemic priming events (e.g. by growth factors, cytokines, immune cells, exosomes secreted by, e.g., primary cancer cells), niche formation (perivascular niches, metastatic niches) [54], specific organ microenvironments and micro environmental changes, and stromal progression as already introduced by Sleeman et al. [40]. Further hypotheses like “self-seeding” as a feedback of tumor cells from metastatic lesions, re-priming the primary tumor, cannot be excluded and are also shown.

crucial role in the development of CSCs, one could speculate that at least some of the cells giving rise to the metastases cells had MetSCs features indeed. Along these lines, it was shown that miR-199 mediated FOXP2 repression equips breast cancer cells with CSC-like traits, increasing not only their tumour-initiating capabilities, but also their metastatic abilities at the same time [59]. Another study has shown that miR-150 altered the expression of more than 30 genes related to CSC signatures, amongst them CBL [60]. Similarly, Tröschel et al. found that BRCA2 is decreased after overexpression of miR-142-3p, leading to the loss of breast cancer stem cell characteristics accompanied by a diminished expression of cluster of differentiation (CD) 44, CD133, aldehyde dehydrogenase 1 (ALDH1) and biorientation of chromosomes in cell division protein 1 (BOD1), which are all associated with cellular stemness [61]. Additionally, Tang et al. has demonstrated that AKT2 serves as a direct target for miR-612, through which epithelial-to-mesenchymal transition (EMT) and stemness in hepatocellular carcinoma are suppressed [62]. Also, the closely related stem cell regulatory gene AKT3 was shown to be regulated at the translational level by miR-93

expression of the stem cell specific transcription factor, achaete-scute homolog 2 (ASCL2) and the insulin-like growth factor 2 (IGF2), leading to higher expression of genes like SRY-box 9 (SOX9) and olfactomedin 4 (OLFM4), also known to be implicated in the presence of CSCs in tumours [57,58] (Fig. 2). In our paper, we found a higher mutational rate in the metastatic lesion of colorectal cancer compared to the matched tumours, leading to the conclusion that after truncal separation individual cell clones evolved [8]. Moreover, the number of somatic mutations found exclusively in the metastases provided a clear hint that disseminated cell clones, through the accumulation of, e.g., genetic aberrations after evading the primary tumour, contribute to the metastatic lesions [8]. In this context, the presence of metastasis specific and recurrently mutated 3′-untranslated region (3´-UTRs) was interesting which affected different genes responsible for stemness and self –renewal features, e.g., forkhead box protein P2 (FOXP2), casitas B-lineage lymphoma (CBL), BRCA2, and protein kinase B (PKB), also known as AKT. Due to the fact that a deregulation of these proteins by microRNAs (miRs) might play a 4

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Fig. 2. CSCs and the origin of metastatically relevant cells or MetSCs: Within the primary tumor, CSCs form a specific subset of cells being the only cell type with long-term self-renewal potential. The induction of stemness features and conversion of non-CSCs to CSCs can be triggered here by multiple genetic and molecular aberrations. CSCs and non-CSCs can be part of the invasive front of the tumor and continuously intravasate into the blood stream, circulate in the body and extravasate to distant sites. Most of those disseminated cells die. The point in time/stage at which MetSCs arise is still unknown and in our opinion flexible, depending on the occurrence of particular genetic/molecular events and environmental interactions. Potential factors contributing to this as discussed in our text are mentioned. Surviving MetSCs and DTCs can enter dormancy at specific sites or niches and can last there for several years. Upon reactivation, those cells can release metastatic progeny into the circulation to initiate metastases in appropriate organs, or maybe even feed back to the primary tumor. Moreover, DTCs are potentially converted into MetSCs by genetic and molecular aberrations during the dissemination and environmental interaction.

within the NOTCH 2 gene that had an activating effect for lymphoid malignancies [66,67]. Similar oncogenic effects caused by NOTCH 1 gain of function mutations were observed in head and neck cancer [68,69]. In contrast to that, loss of function mutations affecting NOTCH family and pathway genes have been described for skin and lung cancers [70], which were supported by studies suggesting a tumor-suppresor role of NOTCH in hepatocellular carcinoma, pancreatic carcinoma and neuroblastoma [71–73]. Here, certainly more detailed investigations must follow which elucidate the specific functional roles of particular mutations within NOTCH genes, or of completely different ways of regulating NOTCH function, with regard to stemness, or for the development of metastatic stem cells. Interestingly, in this context it was shown that NOTCH plays a crucial role to supply the perivascular niche to enable the metastatic outgrowth of cells [74]. Additionally, it was described recently that u-PAR plays a crucial role for the activation of NOTCH 1. It was shown that u-PAR is able to

and miR-659-3p in malignant breast stem cells and neuroblastomas [63,64]. Taken together, the metastasis-specific mutations affecting 3′UTRs of stemness genes we found in metastases of colorectal cancers [8] might represent additional mechanisms to modify miR-regulation of these stemness genes, and we can only speculate that this might assist the development of MetSCs. Certainly, this would need to be investigated in future functional studies and appropriate models [65]. The fact that we found an enriched mutational signature within genes connected to PI3K-AKT signalling, including neurogenic locus notch homolog protein (NOTCH), is interesting in the stemness context because this pathway has been described to be important for the selfrenewal and differentiation of CSCs. However, it remains to be investigated what putative functional roles mutations enriched within NOTCH play for the existence of CSCs or MetSCs. Studies about NOTCH have shown an ambivalent behaviour in the development of various cancers. In this regard, a study by Kiel et al. identified 26 mutations 5

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Furthermore, already in 1999 it has been shown by Gary Gallick’s group that liver metastases, as compared to corresponding primary colorectal cancers, have a significantly elevated endogenous SRC-activity [90–92], and that high SRC-activity is prognostic in colorectal cancer [92]. Although we could not measure SRC activity in the very same hepatic metastasis samples as we used for our recent metastasis genome study, these reports together with an enhanced mutational frequency within the PI3K-AKT axis (and also IGF1) in liver metastases makes a similar importance of SRC/PI3K-AKT activation for colorectal liver metastasis quite likely. The mechanisms leading to high endogenous SRC-activity especially in or during metastasis still remain to be fully investigated, especially since a direct activation via mutations within the SRC molecule, which also were not found in our present genome study, have rarely been reported. It has been hypothesized repeatedly in the past that SRC-activation might happen indirectly via activating pathways or molecules modulating SRC activity [90,91], and in this context it is interesting to note that in our recent whole genome sequencing analysis in colorectal cancer we noticed mutations within the 3′UTR-regions of CBL/ Cbl proto-oncogene B (CBLB) molecules, which can interfere with SRC-activity [93–96], amongst multiple other functions (see below). In line with our present finding in the genomes that mutations were enriched amongst pathways capable of extracellular matrix remodelling and cell adhesion [8], it has been reported that markers characterizing EMT, together with stem cell markers (see chapter above), are overexpressed in disseminated tumor cells from metastasized cancer patients [54,97–99]. Oskarsson et al. outlined that EMT, and the reverse process mesenchymal to epithelial transition (MET), must be critical features of metastasis-initiating cells or “metastatic stem cells” (see chapter above) to disseminate, extravasate and be able to form colonies at the metastatic target organ site. Interestingly, in our earlier systematic study in which we tried to characterize the metastatically relevant miR landscape, again by investigating resected metastases as compared to their corresponding primary colorectal cancers [9], we found that, amongst other miRs, miR-135b, miR-210, miR-218, the latter acting synergistically to the miR-200 family, were not only among the group of most significantly deregulated miRs in the hepatic metastases, but they were found to orchestrate a whole network of target mRNAs among them being E-cadherin, zinc finger E-box-binding homeobox (ZEB)-transcription factors, forkhead Box N3 (FOXN3), SET domain containing 2 (SETD2) which regulated EMT/MET, and by this invasion and metastasis. This observation adds to the picture that EMT/ MET-orchestrating processes are essential for metastasis, specifically metastasis of colorectal cancer to the liver. It is highly interesting in this context that, in our present study on the whole genomes [8], we found several copy number changes which were associated with the deregulated expression of many further miRs in the hepatic metastases we had described previously [9], including, for example, the miR-34 family, miR-30e, miR-122, miR-379, miR-483, and others. Especially miR-122 is of interest in this context, because this miR plays an essential role in maintaining liver homeostasis, and its loss of function is linked to hepatic carcinogenesis [100,101]. This is supported by the fact that this miR plays a key role in preserving the steady-state of the adult liver physiology by suppressing non-hepatic genes, and to maintain a hepatic gene expression signature [102]. These data are challenged by a recent study from Vychytilova-Faltejskova et al. using genome-wide microRNA expression profiling in primary tumors and matched liver metastasis, which revealed, amongst others, an increase in the expression of miR-122 in the tissue of liver metastases [103]. However, we have preliminary evidence that miR-122 might be secreted from liver cells to be taken up by cancer cells in a paracrine fashion (unpublished data, data not shown), which could reconcile both observations. Nevertheless, our aforementioned examples give one genomic explanation for the significant change of expression of particular miRs during metastasis and site-specific metastasis to the liver. Another highly interesting observation of our recent genome study certainly is

prevent the activation of NOTCH by sequestering tumor necrosis factor alpha-converting enzyme (TACE) within lipid rafts of tumor cells [75]. This is interesting because Oskarsson et al. [54], amongst other molecules, highlighted the u-PAR as one molecule which could be an essential characteristic of MeSCs. In our genome profiles of primary colorectal cancers and metastases, we did not find evidence for mutations or significant genomic changes affecting the u-PAR gene [8], however, this matches early previous publications who had consented that u-PAR gene expression is mainly regulated at the transcriptional level, with additional components affecting translation and interactions of the protein with ligands [34,37,76–86], rather than by mutations of the gene. Nevertheless, our group already had suggested the expression of u-PAR as one characteristic of metastatically relevant phenotypes of disseminated tumor cells in solid cancers in 1995 and 1997 [34,37], since we observed disseminated tumor cells expressing u-PAR to undergo a positive selection during follow-up after curative tumor resection of gastric cancers, which correlated significantly with later disease recurrence and metastasis [37]. In the meantime, it has been well documented that the u-PAR, depending on particular ligand interactions such as specific integrin and fibronectin, can be one of the molecules providing a switch between tumor cell proliferation and dormancy [87]. This renders the u-PAR to be a highly likely candidate for being able to keep single disseminated tumor cells in dormancy over years, and reactivating them for outgrowth to a metastasis even years later [32,41]. Thus, the u-PAR might even contribute to the development of MetSCs, especially when conditions of the microenvironment are supportive with, for example, an enrichment in fibronectin, which is the case in bone marrow or the liver, an aspect potentially being important also for site-specific metastasis (see below). Taken together, examples like NOTCH or u-PAR clearly show that looking at genomic, or also epigenetic, changes will certainly not be sufficient to achieve a complete picture of the “metastasome”, and that systematic –omic studies need to be seconded by detailed analyses at the expression and functional level. However, recent genome studies, including ours, can clearly support hypotheses, corroborate changes within genes, and help to clarify if a “metastatic stem cell” might arise from a CSC in a hierarchical mode. 4. Site–specific metastasis of colorectal cancer cells: evidence from the metastasome In our recent work which (with one exception) sequenced the whole genomes of metastases to the liver of colorectal cancers, we found that mutations specific to the metastasis are enriched in mutations within PI3K-AKT pathway molecules, cell adhesion programs, molecules involved in extracellular matrix remodeling, and especially also hepatic stellate cell activation programs. This clearly suggests that, during the evolution of the metastatic clone(s) capable of colonizing the liver, programs for site-specific metastasis are being activated in these cells to enhance their capacity of establishing metastasis in this organ. Earlier reports on colorectal cancer have already outlined the importance of an activation of the PI3K-AKT pathway in colorectal cancer progression in general, in part by mutations [54,88], which is in line with our findings. However, of course genomic changes such as mutations within pathway molecules most certainly are not the only causes for an increased activation of a pathway during cancer progression or metastasis to specific sites. For example, reports by Zhang et al. [54,89] suggested that in bone metastasis of breast cancer, CeXeC motif chemokine 12 (CXCL12)/ stromal cell-derived factor 1 (SDF1) and IGF1 generated by a stroma rich in cancer-associated fibroblasts (CAFs) positively select tumor cell clones with elevated proto-oncogene tyrosine-protein kinase sarcoma (SRC)-activity, which in turn leads to PI3K-AKT pathway activation and increased survival. We speculate that a comparable mechanism might not only hold true for bone metastasis of breast cancer, but also for hepatic metastasis of colorectal cancer, since we also describe IGF1 mutations in hepatic metastases in our paper [8]. 6

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reflected in lung metastases of colorectal cancer [21]).

that we found evidence for genomic changes within 3′UTR sequences of different genes able to modify the metastatic microenvironment, e.g. an immunosuppressive microenvironment (see below), which predicted a putative change of the quality of miR binding to these 3′UTRs [8]. These data need to be investigated closely in functional studies during the upcoming years. Taken together, the data re-emphasizes the diverse role of miRs in metastasis, in the colonization of particular organs like the liver, in priming metastatic niches by being deregulated in, and exerting their molecular interactions by, metastasizing cancer cells, but also in systemic priming activities initiated by primary tumor cells. This can happen via, e.g., the shedding of miRs in exosomes into the systemic circulation as demonstrated before by other groups [54,104,105], and as visualized at the single-cell, single molecule level by an interdisciplinary team effort by us and others recently [106,107]. In the context of exosomes, it is a highly interesting finding that particular integrins harboured by exosomes, but also disseminated tumor cells, can act as “docking partners” to assist in the targeting of particular metastatic niches and organ sites such as the liver [108]. Using exosome proteomics, Hoshino et al. defined a distinct integrin expression pattern connected to lung or liver metastasis. In detail, they demonstrated that the existence of α6β4 and α6β1 integrins was associated with the occurrence of lung metastasis, while αvβ5 integrins found in exosomes redirected the cells to lung and liver. Moreover, this work again showed that integrin uptake by resident cells induced SRC activation, which led to inflammation at this site [108]. This re-emphasizes the importance of an increased SRC-activity for hepatic metastasis of colorectal cancer [90]. Our finding that hepatic stellate cell activation programs appeared activated, as suggested by an enrichment of metastasis-specific mutations within molecules of genes within stellate cell activation programs, in colorectal cancer liver metastases [8], is of particular interest and again supports hypotheses of the activation of site-specific colonization programs in metastasizing cancer cells. As summarized by Erez [109], also for hepatic metastases of pancreatic ductal adenocarcinoma (PDAC) evidence is accumulating that hepatic stellate cells appear to be reprogrammed into myofibroblasts which prime the hepatic microenvironment to a supportive niche for metastasizing tumor cells. Mechanisms leading to this reprogramming of hepatic stellate cells include a recruitment of “metastasis-associated macrophages” (MAMs) from the bone marrow by liver micro metastases [110] and an activation of hepatic stellate cells into myofibroblasts by MAM-secreted granulin. This results in a significant change of the secretome of the fibroblasts, a remodelling of the extracellular matrix (ECM) and especially an up regulation of periostin expression, which has been observed previously to be upregulated in the stroma of metastases of different cancer types, including the colon [111–113]. In pancreatic cancer metastasis to the liver, periostin was functionally required for enhancing metastatic cancer cell growth in PDAC in vitro, as were secreted factors of the hepatic stellate cells in this work [110,112]. This clearly suggested that metastasizing PDAC cells are able to recruit granulin-secreting macrophages systemically from the bone marrow to reprogram hepatic stellate cells to form a (pre-) metastatic niche, at least in part by increasing periostin expression and –activity. Periostin, as SRC in PI3K-AKT activation (see above), and other factors can also assist “metastatic stem cells” to respond to stromal Wnt and NOTCH ligands, this activating self-renewal pathways [54]. It is highly interesting in this context that, in studying the whole genomes of colorectal liver metastases [8], we found several genetic changes for genes involved in hepatic activation pathways, like FAT atypical cadherin 1 (FAT1), fibroblast growth factor 1 (FGF1), kinase insert domain receptor (KDR), or again AKT [114–117]. It remains to be investigated in future years how metastases to different organs will exhibit specific signatures for particular other organs sites, e.g., the brain or the lungs, and whether they will partially overlap with signatures characterizing particular routes of metastatic dissemination, e.g., hematogeneous dissemination via the portal vein (in case of colorectal metastasis to the liver) versus lymphatic spread (which might be

5. The Vogelstein sequence of colorectal cancer progression: new elements as suggested by the metastasome In 1993, Vogelstein et al. [55] already postulated a colorectal cancer progression model suggesting sequential mutational gains within Wnt-, Ras-.the transforming growth factor-β (TGFβ)- and TP53-signalling. Numerous studies in the meantime have shown the functional relevance of the deregulation of these molecules and pathways for the progression and metastasis of colorectal cancer [118–126]. It is amazing that large genome studies such as TCGA, Giannakis et al., and Yaeger et al. [1,6,127] could basically confirm this sequence. Also, our recent whole genome study comparing colorectal primary tumors with corresponding metastases confirms the “Vogelstein-sequence” [8], as do other studies like Brannon et al., or Vermaat et al. [14,26]. These studies as well as ours found a high level of concordance of mutations in known driver genes of tumor evolution and progression such as adenomatous polyposis coli (APC), KRAS proto-oncogene, GTPase (KRAS), TP53 [1,6,8,26,127] and also overlap in some mutations found in transcription factor 7 like 2 (TCF7L2), F-box and WD repeat domain containing 7 (FBXW7), or SOX9 [1,6,127]. Still, with our recent study we could suggest additional components to this sequence as discussed there in detail, or add more information on their exact placement [8]. For example, in our study, SOX9 mutations were found in diploid samples only, which matches to the observation in the TCGA cohort that SOX9 mutations were mutually exclusive with TP53 mutations, which correlate with aneuploidy in our study [2,8]. Instead of mutations found, for example, in the Brannon study within SMAD Family Member 4 (SMAD4) of the TGFβ-pathway [26], our study observed a partial loss on chromosome 18 which contains SMAD2 and 4 genes [8], and this adds to the information on how molecules of this pathway might be modified during colorectal cancer progression. We also found mutations in LDL receptor related protein 1B (LRP1B) (associated with triploid samples), a suppressor of Wnt signaling that has been reported to be especially downregulated in cancers of the right hemicolon [8], in addition to mutated LDL receptor related protein 5 (LRP5) shown in the TCGA cohort [2]. In addition, interesting non-coding mutational events might add to the progression model and should be investigated functionally in future experimental studies. For example, AC010091.1 which we found mutated in about one in four of samples investigated (mutually exclusive with KRAS and TCF7L2 mutations), could potentially act as a decoy for miRs which target FAT atypical cadherin 4 (FAT4) which suppresses the Wnt pathway, thus potentially interfering with the nuclear regulation of β-catenin [8]. Mutations within AC010091.1 could also be confirmed when we searched in the TCGA and other large databases [1,6,127], as could be other non-coding mutations in, e.g., small nucleolar RNA host gene 14 (SNHG14), SRY (sex determining region Y)-box 2 (SOX2−OT), long QT intronic transcript 1 (KCNQ1OT1), interferon-induced very large GTPase 1(GVINP1), hect domain and RLD 2 pseudogene 3 (HERC2P3) and IncRNA AC016683.6 we described [8]. As a further interesting observation within non-coding sequences (proposing further experimental studies in the future), we found that the 3′ UTRs of, e.g., FOXP2, XK Related 4 (XKR4), dachshund family transcription factor 2 (DACH2), ring finger protein 217 (RNF217), matrix metallopeptidase 16 (MMP16), neurotrophic tyrosine kinase, receptor, type 3 (NTRK3), protein prenyltransferase alpha subunit repeat containing 1 (PTAR1) and zinc finger protein 793 (ZNF793) were mutated in our cohort [8] and this could also be seen when we searched other large colorectal cancer sequencing databases [e.g. [1,6,127],]. Also, mutually exclusive to 3′-UTR-mutated XKR4, we observed 3′UTR mutations in anoctamin 5 (ANO5) [8] and both XKR4 and ANO5 have been suggested to be able to participate in the externalization of phosphatidyl serine, and therefore in the creation of an immunosuppressive environment [8,128]. Generally speaking, it has been suggested that metastatic progression, and the “metastatic switch”, 7

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hepatic metastases as compared to corresponding primary tumors (see below), which was supported by later work of Kim et al. [12]. Furthermore, in a recent single cell sequencing study at 2 colorectal cancer patients in which metastases and primary tumors were analysed [13], these colleagues found evidence for the presence of particular metastasis-specific mutations, however, even with performing additional active targeted ultra-deep re-sequencing within the corresponding primary tumor, they could not find these within the primary, concluding that these mutations must have developed after clonal separation of the metastasis from the primary tumor. Therefore, from a clinical perspective we think that such findings are absolutely relevant and important for principal future diagnostic and therapeutic considerations, regardless of potential additional methodological explanations. If, e.g. in 12 of 12 cases as in our study, genomic lesions specific for the metastasis would not have been detected, or detectable, even in whole genome sequencing of the primary tumor of cancer patients with latest sequencing technology, chances are high that even in the most timely personalized strategies of the internationally leading Comprehensive Cancer Centers (CCCs) which match primary and first line, including targeted, therapy to the genome of the individual primary tumor, there are still considerable chances to therapeutically miss the metastatically relevant tumor cell clones, thus missing the chance to prevent later metastatic disease. This certainly might be one explanation for the unchanged situation that metastases still arise after complete therapeutic elimination of the primary tumor and, even in the modern era of personalized medicine, still lead to about 90% of deaths related to cancer. This certainly calls for diagnostic, therapeutic and preventive consequences for the clinic. First, we must initiate concerted programs in which genetic, epigenetic and molecular findings specifically detected in metastases enter early diagnostic strategies, ideally before macroscopic metastasis will be detected. In the era of “radiomics” and latest microscopic imaging, for example super-resolution technologies which can detect single molecules at the single cell level with resolutions down to 5 nm [132], and given continuously ongoing exciting developments in live cell imaging which can differentiate, e.g., different modes of migration patterns of tumor cells which might warrant individual therapeutic consequences [133–136], initiatives must be launched which combine micro- with macroscopic imaging to a synergistic effort. For example, a rapid development of novel molecular tracers that are capable of visualizing genomic changes which have been identified specifically in metastases could be introduced into cancer patient diagnostics, and anticipated easily for modern radiology and imaging departments. Technically, it has been shown already that the detection of such genomic lesions in principle can be done at the level of chromosomal aberrations, gene copy number changes (e.g., EGFR amplifications), even single mutations, etc., with different methodologies [137–141]. Also, even the subcellular distribution patterns of particular molecules critical for metastasis, e.g. particular miRs in compartments like exosomes, could be useful for the detection of metastatically relevant tumor cell populations [106] within a heterogeneous scenario such as a primary tumor. Second, we must seek to include therapeutic strategies which are targeted against genomic, epigenetic, and other molecular changes found primarily in metastases into early therapeutic concepts, with a long-term objective to prevent metastases, applying stepwise clinical study concepts seconded by specific translational investigations. This could even include not only metastasis-specific findings, but also novel biomarkers found in the primary tumors and metastasis if they have implications for both regarding, e.g., decision making for particular types of therapy. For example, if in experimental and larger translational-clinical studies it would be confirmed that ARHGEF7/33 mutations as suggested by us [8], but as we also found them in the databases of other large studies in primary tumors [1,127], are mutually exclusive with K- and N-RAS-mutations [8], and if ARHGEF mutations would exert a similar function in EGFR-based therapy as RAS- mutations as we

certainly also is associated with modifications of the immune microenvironment as one component of stromal progression [129,130], and this can be further supported by actual findings of genomic changes concerning molecules being able to modulate immune evasion. In this context, further interesting genomic findings of our recent work are 3′UTR mutations within CBL/CBLB [8]. CBL, an E3 ubiquitin protein ligase, and also in part its paralogue CBLB, has been reported to exert multiple functions such as the nuclear degradation of β–catenin [8], the downregulation of programmed death-ligand 1 (PD-L1), the negative regulation of epidermal growth factor receptor (EGFR)-signalling, but also the activity and signalling of SRC-kinase family members (see above). Thus, these molecules, and mutations modulating their expression or activity, might play interesting roles in immune evasion, colorectal cancer progression and metastasis, although exact functions and molecular mechanisms still need to be investigated in detail. One of the most interesting findings adding to the “Vogelstein-sequence” was of course the discovery of Rho guanine nucleotide exchange factors (ARHGEF) mutations in our recent study, ARHGEFs being guanine nucleotide exchange factors which facilitate small GTPases such as rat sarcoma (RAS)-family members [131]. In this context it is of particular interest that we observed an exclusivity of AHRGEF 7- and -33 mutations with KRAS- and NRAS proto-oncogene, GTPase (NRAS)-mutations [8]. Mutations in ARHGEF genes were also found in other studies [1,6,127], especially within RhoGEF- and plekstrin homology domains [1,127]. Moreover, we found that ARHGEF7 mutations exhibiting mutual exclusivity to KRAS mutations in our and the TCGA study [1,8], are associated with worse disease-free survival (p = 0.004), and that patients with ARHGEF mutations of any kind generally exhibited worse disease-free survival (p = 0.04). Due to these observations, we speculate that ARHGEF mutations might have a similar function as RAS-mutations within EGFR-based therapy, and this hypothesis should be tested as soon as possible in first clinical studies (see below). Taken together, it is amazing that the classical Vogelstein-model of colorectal cancer progression can be confirmed in all actual genome studies, which now can be further refined by discoveries as described above and as graphically summarized in Fig. 7 of our recent original article [8]. It will be interesting to functionally investigate the exact molecular actions and interaction of several novel components as suggested. 6. Translational and clinical consequences as suggested from the metastasome In our recent whole genome analysis, we found evidence that the metastases harbour genomic changes (e.g., 19% of all SNVs/single small mutations detected in our study) that are private to the metastases and not being shared with the primary tumor. This was seen in all of the 12 cases of paired metastasis and corresponding primary colorectal cancer samples we had the opportunity to investigate [8]. Certainly, one explanation for this observation could also be that the sample of the primary tumor taken for the genome analysis did not include the clone(s) which gave rise to the later metastasis and therefore, the genomic lesions seen specifically in the metastasis were not detectable in the corresponding primary tumor sample. Another methodological explanation might be that the cancer cell population giving rise to the metastasis was present in the corresponding primary tumor sample, but in cell numbers too small for being detectable within the background of the genetic signature of the other sub clones. Such methodological explanations cannot be excluded, as discussed earlier by others (e.g., Turajlic and Swanton, Kim et al. [12,46]), and can certainly also hold true for our study. Still, however, other studies support the findings of genomic changes private to the metastases. For example, Vermaat et al. [14], in targeted exon sequencing of formalinfixed paraffin-embedded (FFPE) samples in 21 metastasized colorectal cancer patients, had clearly seen considerable genomic changes in 8

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sarcoma (FUS), PIK2CG, helicase with zinc finger (HELZ), spen family transcriptional repressor (SPEN), death associated protein kinase 1 (DAPK1), zinc finger protein 521 (ZNF521), RNA binding fox-1 homolog 1 (RBFOX1), transformation/transcription domain associated protein (TRRAP), GATA binding protein 1 (GATA1), and others. In addition, the latest publication of Kim et al. [12,145] also observed metastasis-specific small mutations and other genomic changes with whole-exome sequencing, some of them like APC-mutations or copy number changes/ structural variations concerning mono-ADP ribosylhydrolase 2 (MACROD2), fragile histidine triad diadenosine triphosphatase (FHIT), and parkin RBR E3 ubiquitin protein ligase (PARK2) overlapping with our findings [8], some others like mutations within NEDD4 like E3 ubiquitin protein ligase (NEDD4L) not overlapping. Still, the discovery that NEDD4L is able to interact with SMAD proteins, thus regulating TGFβ signaling in the metastasis context [146,147], adds to our observation of losses of SMAD2 and SMAD4 on chromosome 18 in hepatic metastasis in terms of the general observation of genomic changes within the TGFβ-pathway during colorectal cancer progression [8,148]. Still, although some findings of our paper, the paper of Leung et al., Brannon et al., Vermaat et al., our paper [8,13,14,26], and others are overlapping, especially regarding the gross mutational observations within colorectal cancer progression pathways like Ras, Wnt, PI3K, APC, TGFβ, etc., of course it is a little concerning that some observations of metastasis-specific genomic lesions, especially regarding single mutations, have been made rather in single or a few cases in each of these studies, due to the general drawback that most centers could study, and have available, only a few cases of paired metastasis and corresponding primary tumor tissues. This underscores the urgent importance of initiating concerted, world-wide multicenter efforts together, to be able to investigate frequencies of particular mutations and further genomic lesions within really large numbers of resected metastases of cancer patients, and to discover more gross patterns across several hundreds of metastases as a true advancement of a therapeutic concerted action against metastases. Still, summarizing all the aforementioned data and discussions, we think that some findings resulting from “metastasome” studies in colorectal cancers could be put forward already for first early clinical trials accompanied by translational studies which, e.g., might measure particular genomic, and certainly also other molecular, changes in biopsies of metastases of individual colorectal cancer patients participating in these. For example, a study measuring ARHGEF mutations and correlating them with the clinical response of metastasized RAS-mutationnegative patients to EGFR-based therapeutics could be launched immediately. Also, heading for immediate multicenter descriptive studies on the frequency of particular mutations at hundreds of metastases within targetable molecules, as suggested by others and our latest study, could serve as a basis for future clinical studies on targeted therapeutics tailored to mutations detected at higher frequencies. Moreover, findings which definitely overlap between our present study and others [6,12,14,26], for example on a higher mutational frequency within the PI3K-AKT pathway in hepatic metastases (see above and chapter site-specific metastasis), in our opinion could already be translated into first clinical trials on existing PI3K-AKT inhibitors at (metastasizing) colorectal cancer, paralleled by translational research programs investigating such mutations at primary tumor/metastasis tissue biopsies of participating patients. In parallel to such programs, liquid biopsies could be taken to investigate the sensitivity or specificity achievable with more easily accessible patient samples for future studies. Certainly, also discoveries made many years back on, e.g., a higher endogenous SRC-activity within liver metastases of colorectal cancer [90] could easily lead to clinical studies that investigate SRC-inhibitors like dasatinib as tools against liver metastases of colorectal cancer [149,150], especially since latest data put forward hypotheses on a potentially deeper understanding on SRC/SRC-family-member-activation (see above) during colorectal cancer metastasis. Also, the

speculate from our data, ARHGEF mutations should be included into the portfolio of novel biomarkers in colorectal cancer that represent a contraindication for EGFR-based targeted therapy in such specific patients. Also, our finding that primary colorectal tumors and metastases share particular mutational patterns such as the AC1 or AC5 age-related patterns as truncal ones, but then develop rather private mutational events within particular patterns such as the AC3 pattern (doublestrand DNA repair/BRCAness patterns), we might speculate that poly ADP ribose polymerase (PARP) inhibitors should be tried as potentially useful therapeutic agents in colorectal cancer in first clinical studies, hypothetically being useful for treating primary tumors as well as metastases of this tumor entity which harbor AC3-mutational patterns and BRCAness signatures [8,142]. In single cases, we found genomic changes which were enriched in the metastases, or found exclusively in the metastases, which concerned already targetable molecules according to actual databases [8]. Specifically, we observed one RASmutation-negative case of a metastasis with 4 copies of an EGFR amplification as compared to the corresponding primary tumor (3 copies), suggesting opportunities for EGFR-based therapies such as Cetuximab [8] in such a metastasized patient, although we found an ARHGEF33 mutation in the same patient in parallel which might again call for caution as explained above. This is in line with one case reported by Brannon et al. [26] who had performed targeted deep coverage sequencing of 230 cancer-related genes, whereby they found high-level EGFR amplifications in the metastasis of one particular colorectal cancer patient but not in the corresponding primary tumor. Interestingly, perfectly in line with our observation in one case, this patient in the Brannon study showed an absence of K- or N-RAS-mutations (also of B-Raf proto-oncogene, serine/threonine kinase (B-RAF)-mutations) in parallel to the EGFR amplification, and it would be interesting to see the ARHGEF mutational status of this patient. Also, in the paper by Vermaat et al. [14], the authors report cases in which, for example, SNVs within the EGFR gene are increased in hepatic metastases as compared to the corresponding primaries, this collectively implicating a trend of increasing genomic instability activities within the EGFR gene during colon cancer metastasis. The Brannon study, having looked at a selected gene panel, also confirms our observations that there are occasional mutations private to the metastasis which are not shared with the corresponding primary tumor, which clearly argue for the importance of analyzing individual metastasis tissue to include such observations into individual therapeutic concepts. Some of them concerned the EGFR-and the phosphoinositide 3-kinase (PI3K)-pathway (which is in line with our observation of increased mutational activities within this pathway in the hepatic metastases, see chapter site-specific metastasis), some concerned mitogen-activated protein kinase (MAPK)-pathway-related molecules, SMAD-genes, or runt-related transcription factor 1 (RUNX), and protein tyrosine kinase 7 (PTK7) and cyclin-dependent kinase 8 (CDK8) as regulators of Wnt-signaling [26,143,144]. Our own finding of metastasis-exclusive mutations within, e.g., FAT1 which modulates EMT and stemness and can induce aberrant Wnt-signaling [8] would be in line with these observations of mutations destabilizing Wnt-related pathways during colorectal cancer metastasis, opening potential avenues for targeted treatment, e.g., with mAB198.3 [8] in metastatic colorectal cancer. Observations by Vermaat et al. [14] of different numbers of SNVs within the vascular endothelial growth factor receptor 1 (FLT1) and the kinase insert domain receptor (KDR) gene are also in line with our observations of mutations within these genes in the sequences of colorectal cancer metastases [8]. This suggests that particular metastatic colorectal cancers could be successfully treated with drugs such as axitinib, sorafenib or cabozantinib which inhibit VEGF-induced activities mediated by the KDR towards, e.g., cell survival, migration, and sprouting [8]. Also, the single-cell sequencing study of Leung et al. [13] identified single mutations private to the metastasis which were not shared with the corresponding primary tumor in 2 colorectal cancer cases, including mutations within fused in sarcoma/translocated in 9

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observation that SRC is an important activator of the expression of metastasis-promoting molecules like u-PAR [78], u-PAR as a switch between tumor cell proliferation and dormancy [87] having been suggested by us already in 1995 to be a phenotypic characteristic of metastatically relevant disseminated tumor cells in gastrointestinal cancers [37], underscores the opinion that it might be worth giving such therapeutics a try in the treatment, or even prevention, of colorectal cancer metastasis.

[7]

[8]

7. Summary and conclusions [9]

In summary, recent exciting developments in the systematic discovery of specific changes characterizing metastases as opposed to corresponding primary tumors at the genome, transcriptome, further –omics and the functional level provide a deeper understanding of metastasis evolution and open new avenues for developing targeted personalized concepts towards metastasis. We have tried to outline this with some perspectives for colorectal cancer in this review, scetching an actual model for metastasis evolution and suggesting some clinical consequences for this tumor entity. Still, to optimally achieve the goal of completely understanding and defining the “metastasome” and achieve concerted metastasis-prevention efforts, a world-wide collaborative initiative between multiple centers is necessary. Only this can enable us to collectively study large numbers of metastases, and also to investigate specific characteristics of metastases of diverse primary tumor origins to particular organ sites, as a systematic hypothesis generation for site-specific metastasis. “Healing is a matter of time, but it is sometimes also a matter of opportunity” (Hippokrates of Kos, 460-370BCE; [Reference https:// brainyquote.com]). — Since metastasis is still the most deleterious killer in cancer diseases, we think the time seems right to initiate a worldwide “human cancer metastasome project” as a systematic and collaborative effort on understanding, combating and preventing metastasis. We hope that this review can stimulate, and invite, as many colleagues of the world as possible to collectively seek the opportunity.

[10]

[11]

[12]

[13]

[14]

[15] [16]

Declaration of Competing Interest [17]

The authors declare that there are no conflicts of interest.

[18]

Acknowledgements

[19]

The work of our group was supported by the Alfried Krupp von Bohlen und Halbach Foundation, Essen, the Deutsche Krebshilfe, Bonn (70112168), the Deutsche Forschungsgemeinschaft (DFG, grant numberAL 465/9-1), the HEiKA Initiative (Karlsruhe Institute of Technology/University of Heidelberg collaborative effort), the HIPO/ POP-Initiative for Personalized Oncology, Heidelberg (H032 and H027) and by the Molecular Biomarkers for Individualized Therapy (MoBIT) project initiative.

[20] [21] [22] [23]

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