Cell Fusion As a Hidden Force in Tumor Progression

Published OnlineFirst November 3, 2009; DOI: 10.1158/0008-5472.CAN-09-2159

Review

Cell Fusion as a Hidden Force in Tumor Progression

Xin Lu and Yibin Kang

Department of Molecular Biology, Princeton University, Princeton, New Jersey

Abstract to poor prognosis and cancer stem cell phenotypes (3). Third, Cell fusion plays an essential role in fertilization, formation of chronic inflammation has recently been shown to dramatically placenta, bone and muscle tissues, immune response, tissue increase the frequency of cell fusion between hematopoietic cells repair, and regeneration. Increasing recognition of cell fusion and various somatic cells such as cardiomyocytes, skeletal mus- in somatic cell dynamics has revitalized the century-old hy- cle, hepatocytes, and Purkinje cells during tissue repair and pothesis that cell fusion may contribute to the initiation and regeneration (9, 10). Similar as-yet-unknown mechanisms under- progression of cancer. In this review, we discuss findings from lying this observation may also stimulate the fusion between bone experimental and clinical studies that suggest a potentially marrow-derived cells (BMDC) and tumor cells, because inflamma- multifaceted involvement of cell fusion in different stages of tion is often associated with the tumor microenvironment. “ ” tumor progression, including aneuploidy and tumor initia- Fourth, cell fusion may be the by-product of cell-eat-cell pro- tion, origin of cancer stem cells, multidrug resistance, and cesses, such as phagocytosis of tumor cells by macrophages (3) the acquisition and diversification of metastatic abilities. or entosis (11). Finally, although rare accidental somatic cell [Cancer Res 2009;69(22):8536–9] fusion almost inevitably leads to cell cycle arrest and terminal differentiation in normal tissues, tumor cell fusions may produce proliferating cells capable of clonal expansion. This possibility Introduction was supported by a recent study showing that fused cells harbor- Cell fusion is believed to be a relatively rare and strictly regulated ing oncogenes E1A and HRAS could override the cell cycle arrest phenomenon that only takes place in specific occasions, such as and evolve to become tumorigenic cells after massive chromo- fertilization (fusion of sperm and egg), the formation of placenta somal rearrangement (7). (fusion of trophoblasts) and muscle fibers (fusion of myoblasts), Cell fusion can be easily recognized in cell culture and animal and bone homeostasis (formation of multinuclear osteoclasts for tumor model experiments using lineage-specific tracking markers. bone resorption; ref. 1). The concept of cell fusion as a mechanism In contrast, fusion events in human cancers are much more diffi- of tumorigenesis dates back to the early 1900s when Otto Aichel cult to detect because of the lack of applicable genetic tracing postulated that spontaneous fusion between somatic cells may lead tools. Therefore, definitive and direct observation of cell fusion to chromosomal abnormalities and cancer (1–4). However, in the in human cancer is rare and the most striking one was reported modern era of molecular oncology, cell fusion has been considered in patients who developed renal-cell carcinoma after allogeneic as an oddity that attracts little attention compared with other bone-marrow transplantation from donors of the opposite gender fields of cancer research. Recent discoveries of a much broader role (12). Karyotyping of the cancer cells showed that they had adopted of cell fusion in tissue homeostasis and regeneration, particularly chromosomes from bone marrow donors, possibly through fusion during inflammatory conditions, have revitalized the interest in cell of early tumor cells with transplanted bone marrow cells. In mul- fusion as one of the driving forces of cancer progression. tiple myeloma cancer patients, up to 30% of the multinuclear os- Spontaneous cell fusion in tissue culture or in animal models teoclasts are found to contain malignant nuclei with typical has been reported for a large variety of tumor cells (1, 5). The fre- chromosomal translocations of myeloma (13). Premature chromo- quency of cell fusion can be up to 1% in vivo in experimental tumor some condensation, a typical result of heterophasic cell fusion, has models (1). Furthermore, fusion efficiency can be proportional to been observed in various types of cancer (1). Binuclear and multi- the malignant level of tumor cells (6). Several reasons may account nuclear cells are also frequently observed in many types of tumors, for the increased fusogenicity of tumor cells. First, enveloped although cell fusion is only one of several mechanisms to generate viruses may trigger the fusion of infected tumor cells (7, 8) and such cells. The correlation between increased DNA content and en- may therefore increase the efficiency of fusion in infection-related hanced malignant behavior has been observed in ovarian cancer, cancers, such as liver cancer or cervical cancer. Second, deregula- prostate cancer, colon carcinoma, and breast cancer (1). Despite tion of physiological fusogenic proteins in tumor cells may pro- the paucity of direct evidence of cell fusion in human cancer, ac- mote cell fusion (7). For example, syncytin, the Env protein of cumulating experimental evidence in recent years has suggested HERV-W human endogenous retroviruses and putative mediator a possible broad involvement of cell fusion during the initiation, of trophoblast fusion, was also found to mediate fusion between progression, and phenotypic diversification of cancer. breast cancer cells and endothelial cells (7). CD44, a cell surface receptor known to be involved in cell fusion during osteoclastogen- esis, is frequently overexpressed in cancer cells and has been linked Cell Fusion in Aneuploidy and Tumor Initiation The direct consequence of fusion between two diploid somatic cells is the generation of a tetraploid hybrid. The nuclei of the fu- Requests for reprints: Yibin Kang, Department of Molecular Biology, Washington sion partners often remain separate, as in the case of cell fusion in Road, LTL 255, Princeton University, Princeton, NJ 08544. Phone: 609-258-8834; Fax: placenta, muscle, and bone. However, in nonprogrammed acciden- 609-258-2340; E-mail: [email protected]. ©2009American Association for Cancer Research. tal fusion, the two nuclei may fuse and lead to aneuploidy and po- doi:10.1158/0008-5472.CAN-09-2159 tentially cancer, given the existence of other genetic alterations,

Cancer Res 2009; 69: (22). November 15, 2009 8536 www.aacrjournals.org

Figure 1. The potential multifaceted role of cell fusion in cancer progression. A, fusion-induced tetraploidy as an intermediate step before aneuploidy (7, 8, 16, 17). Mutations of certain oncogenes (e.g., H-Ras) or tumor suppressor genes (e.g., p53), represented by lightning bolts, in the fusion partners may be required to generate a synkaryonic proliferating cell. CIN, chromosomal instability; n denotes haploidy. B, a biphasic role of supernumerary centrosomes in tumor progression (18). Fusion-induced centrosome amplification causes multipolar division and aneuploidy in nascent tumor cells, when the mechanism(s) for suppressing spindle multipolarity is not activated (“OFF” state). Once the tumor cells acquire the ability to suppress supernumerary centrosomes (“ON” state, e.g., centrosome clustering or orphan centrioles), this mechanism allows the maintenance of a relatively stable chromosomal composition and the clonal expansion of the dominant population. C, generation of a cancer stem cell by having an adult stem cell as a fusion partner (2). Oncogenic mutations could occur in the stem cell, the differentiated cell, or the hybrid. D, fusion between a tumor cell and a BMDC generates a metastatic cell (3). In C and D, reduction division could occur in the hybrid so that the transient increased of ploidy cannot be detected. E, fusion between tumor cells with different metastatic organotropism leads to rapid acquisition of dual-tropisms and may increase dissemination and promote metastatic recurrence after therapy (18). The original single organotropism could be derived from a previous fusion of a tumor cell with an organotropic leukocyte. www.aacrjournals.org 8537 Cancer Res 2009; 69: (22). November 15, 2009

Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst November 3, 2009; DOI: 10.1158/0008-5472.CAN-09-2159 Cancer Research such as loss of p53 function (14). Indeed, tetraploidy frequently initiating cells, that are capable of self-renewal and differentiation precedes aneuploidy in cancer, as was observed in both clinical into diverse tumor cell populations (2). Inspired by the recent find- tumor samples (15) and experimental models (14). A fusion- ings that hematopoietic stem cells are frequently found to be fused mediated oncogenesis model based on these observations has been with several resident tissue cells during tissue repair and regener- proposed (Fig. 1A; refs. 7, 8, 16, 17). When accidental cell fusion or ation, Bjerkvig and colleagues proposed that fusion of an adult cytokinesis failure occurs in a cell with inactivated p53-dependent stem cell (local or hematopoietic) with a differentiated cell may tetraploidy checkpoint (17) or fusion checkpoint (7), supernumer- lead to the formation of a cancer stem cell if either one of the cells ary centrosome-mediated multipolar division, chromosome mis- harbors appropriate mutations to sustain a proliferative fusion segregation, and increased DNA damage rate may then facilitate (Fig. 1C; ref. 2). Several lines of evidence support the feasibility of the progression from tetraploidy to aneuploidy (16). Oncogene ac- this hypothesis. Fusion of BMDCs with differentiated adult tissue tivation and loss of tumor suppressor genes caused by aneuploidy cells was dramatically increased by chronic inflammation, which is in a stochastic manner may select for cell or cells that eventually a major risk factor for tumorigenesis (9, 10). In a mouse model of become fully malignant. It should be noted that the prevailing gastric cancer produced through chronic inflammation from model of aneuploidy focuses on checkpoint mutations and telo- Helicobacter pylori infection, BMDCs were found to replace the mere attrition instead of tetraploidy. The exact contribution of cell damaged epithelial lining of gastric mucosa and eventually gave rise fusion to aneuploidy still awaits further characterization. to gastric cancer (22). Although no direct evidence of cell fusion was The doubling of centrosomes after cell fusion does not always found in this study, additional experiments using fusion tracing lead to chromosome instability. For example, stable chromosome techniques, such as Cre-mediated activation of a LacZ reporter pre- numbers were reported in hybrids resulting from fusion between ceded by a lox-franked stop signal (Cre and LacZ constructs are Hela cells and fibroblasts, between mouse mammary tumor cells, separately integrated in the donor cells and host cells), should be or between ES cells and fibroblasts (18). In a recent study from our used to definitely investigate the potential role of cell fusion in tu- laboratory, spontaneous fusion hybrids between two sublines of mor initiation using this experimental model. the MDA-MB-231 breast cancer cell line were found to have robust chromosomal stability over long-term proliferation in vitro or in vivo (18), despite the fact that more than 50% of hybrid tumor Acquisition of Drug Resistance through Cell Fusion cells have an abnormally high number of centrioles. Further anal- Cell fusion may allow the fused cells to become more resis- ysis revealed that extra centrioles can be discarded, coalesced, or tant to chemotherapeutic drugs in two ways: acquisition of drug orphaned, so that tumor cells were able to maintain bipolar di- resistance from both parental cells and emergence of new drug vision with two functional centrosomes. The ability to control su- resistance. For example, when 5-fluorouracil-resistant and meth- pernumerary centrosomes through such mechanisms is not otrexate-resistant mammary tumor cell lines were fused, the re- specific to breast cancer cells, as similar phenomenon was also sulted hybrids became resistant to both drugs as well as another observed in neuroblastomas (19). The stark contrast between drug, melphalan (1). In a separate study, fusion of a drug-sensitive highly malignant tumor cells and precancerous or early stage tu- E1A-transformed cell with a primary fibroblast generated a hy- mor cells (14), in terms of chromosomal stability after cell fusion, brid that is resistant to drug-induced apoptosis (7). Acquisition suggested a possible biphasic role of centrosome duplication in of new drug resistance may occur at a low frequency and in a tumor progression (Fig. 1B). In the early stage of tumorigenesis, transient manner from a vast variety of the aneuploid progenies centrosome amplification caused by cell fusion, abortive division, resulting from cell fusion. However, even a temporal advantage or abnormal centrosome duplication leads to multipolar division over other tumor cells in drug resistance could allow the escape and chromosome instability, resulting in a heterogeneous pool of of rare cells, which may become the source of tumor relapse (1). aneuploid cells. A random activation of certain intrinsic mechan- As drug treatment of cancer is often accompanied by increased isms for suppressing supernumerary centrosomes in some tumor inflammation, which is known to promote cell fusion, the poten- cells allows the clonal outgrowth of the cells to become the dom- tial contribution of cell fusion to drug resistance should not be inated population with a relatively stable chromosomal composi- underestimated. tion. Indeed, many previous studies have repeatedly shown that the chromosome aberration spectrum stabilizes in advanced cancers (20). The ability to avoid mitotic catastrophe in the face of Cell Fusion and Metastasis supernumerary centrosomes may be an important tumor survival Metastasis is a multistep process requiring the tumor cells to be mechanism that can be exploited for therapeutic intervention. equipped with properties they rarely need in the primary site, in- For example, several proteins have been implicated in the suppres- cluding higher motility, survival in circulation, invasion through sion of spindle multipolarity caused by supernumerary centro- tissue layers, and growth in an unfamiliar microenvironment somes, including NuMA and dynein (21). Targeting these proteins (23). Many of these are intrinsic properties of migratory BMDCs, may lead to catastrophic chromosome mis-segregation and cell such as macrophages. Thus, fusion between tumor cells and death. Importantly, the ability for fusion hybrids to sustain a rela- BMDCs may be an efficient way for rapid acquisition of metastatic tively stable genomic composition will allow tumor cells to acquire phenotypes (Fig. 1D; ref. 3). Indeed, in vitro and in vivo fusion be- and stably maintain additional malignant properties, such as stem- tween tumor cells and macrophages has been shown to produce ness, drug resistance, and metastatic abilities, through cell fusion. hybrids with increased metastatic abilities (3). To test the contribution of cell fusion to the natural development of metastasis, fusion of transplanted bone marrow cells with resident tumor cells Cell Fusion as an Origin of Cancer Stem Cells should be distinguished from unfused tumor cells in mouse tumor Increasing evidence suggests that tumor growth and recurrence models using reporter genes (e.g., Cre-activated LacZ expression). relies on the existence of cancer stem cells, also known as tumor- Examination of nascent distant metastases may reveal whether

Cancer Res 2009; 69: (22). November 15, 2009 8538 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 5, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst November 3, 2009; DOI: 10.1158/0008-5472.CAN-09-2159 Cell Fusion in Tumor Progression fusion between tumor cells with BMDCs may play a significant may increase the efficiency of metastatic dissemination because role in promoting metastasis. the hybrids are able to colonize several different organs when they The organ that tumor cells prefer to colonize, or organotropism, are in circulation, although tumor cells with only a single metasta- can also be influenced by cell fusion. For example, fusion of mye- sis organotropism can only colonize one organ and will be lost loma cells with B lymphocytes resulted in a hybrid cell metastasiz- when they are lodged into an inhospitable microenvironment. Fur- ing to the spleen and liver, yet fusion with macrophages led to thermore, because metastases in different organs often have differ- metastasis to lung (24). Importantly, fusion of tumor cells with res- ent responses to therapeutic treatments, tumor cells with multiple ident cells in a secondary organ may allow disseminated tumor metastasis organotropisms can survive in a different organ and cells to survive in a hostile microenvironment as minimal residual then quickly recolonize the organ that has positively responded diseases and emerge as overt metastasis after accumulation of ad- to therapies. ditional oncogenic alternations. In a recent study, untransformed Overall, accumulating evidence has suggested a plausible in- mammary cells were found to persist in the lung as small clusters volvement of cell fusion in several aspects of cancer progression. until inducible activation of oncogenes stimulated the formation of Rigorous genetic studies in animal tumor models will be needed to pulmonary metastases (25). It is conceivable that fusion between definitely show the extent of cell fusion to tumor initiation, drug the disseminated mammary cells and the local resident cells in resistance, and metastasis. Such experiments, together with more lung, including lung epithelial cells and recruited BMDCs, may al- extensive investigations of cell fusion in human cancer patients, low the survival of disseminated nontransformed mammary epi- may lead to novel therapeutics for cancer. thelial cells and contribute to the formation of metastases when additional oncogenes are activated. Again, such a hypothesis could be tested definitively by using fusion-dependent activation of re- Disclosure of Potential Conflicts of Interest porters in transgenic animal models. No potential conflicts of interest were disclosed. Fusion between tumor cells in a heterogeneous population, such as pleural effusions, may contribute to the rapid acquisition of multiple metastasis organotropisms. In vivo selection of the Acknowledgments MDA-MB-231 breast cancer cell line in mouse models successfully Received 6/11/09; revised 7/30/09; accepted 8/5/09; published OnlineFirst 11/3/09. derived bone- and lung-tropic sublines with distinct organ-specific Grant support: Y. Kang is a Champalimaud Investigator and a Department of metastasis gene signatures (23). In our recent study, spontaneous Defense Era of Hope Scholar Award recipient. Research in the authors' laboratory in vitro in vivo is additionally supported by a research scholar grant from the American Cancer fusion or between the bone-tropic and the lung- Society, the National Institutes of Health, and the New Jersey Commission on Can- tropic subpopulations resulted in stable hybrids with dual trop- cer Research. X. Lu is a recipient of a Harold W. Dodds Fellowship from Princeton isms to both organs (Fig. 1E; ref. 18). Expression analysis showed University. We thank members of our laboratory for helpful discussions. We also apologize to that the hybrids co-expressed both organ-specific metastasis gene the many investigators whose important studies could not be cited directly here signatures. Hybrid cells with multiple metastasis organotropisms owing to space limitations.

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www.aacrjournals.org 8539 Cancer Res 2009; 69: (22). November 15, 2009

Cell Fusion as a Hidden Force in Tumor Progression

Xin Lu and Yibin Kang

Cancer Res 2009;69:8536-8539. Published OnlineFirst November 3, 2009.

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