Acquisition of cancer stem cell capacities after spontaneous cell fusion.

BACKGROUND: Cancer stem/Initiating cell (CS/IC) hypothesis argues that CS/ICs are responsible of tumour initiation, drug resistance, metastasis or disease relapse. Their detection in several cancers supports this concept. However, their origin is still misunderstood. Cell fusion is shown to take part in the formation of CS/ICs, i.e. fusion between mesenchymal stem cell and cancer cell. In a previous paper, we described that fusion leads to hybrids with metastatic capacity. This process triggered genomic rearrangements in hybrid cells together with increased metastasis development. Here, we hypothesize that cell fusion could be strong enough to provoke a cellular reprogramming and the acquisition of CS/IC properties, promoting metastasis formation. METHODS: After spontaneous cell fusion between E6E7 (IMR90 with the oncogenes E6 and E7) and RST (IMR90 fully transformed) cell lines, hybrid cells were selected by dual antibiotic selection. Cancer stem cells capacities were evaluated regarding capacity to form spheres, expression of stem cell markers and the presence of ALDHhigh cells. RESULTS: Our data show that after cell fusion, all hybrids contain a percentage of cells with CS/ICs properties, regarding. Importantly, we lastly showed that NANOG inhibition in H1 hybrid decreases this migration capacity while having no effect on the corresponding parental cells. CONCLUSIONS: Altogether these results indicate that the combination of CS/ICs properties and genomic rearrangement in hybrids is likely to be key to tumour progression.

Cell fusion enhances energy metabolism of mesenchymal tumor hybrid cells to sustain their proliferation and invasion.

BACKGROUND: Cell-to-cell fusion is emerging as a key element of the metastatic process in various cancer types. We recently showed that hybrids made from the spontaneous merging of pre-malignant (IMR90 E6E7, i.e. E6E7) and malignant (IMR90 E6E7 RST, i.e. RST) mesenchymal cells recapitulate the main features of human undifferentiated pleomorphic sarcoma (UPS), with a highly rearranged genome and increased spreading capacities. To better characterize the intrinsic properties of these hybrids, we investigated here their metabolic energy profile compared to their parents. RESULTS: Our results unveiled that hybrids harbored a Warburg-like metabolism, like their RST counterparts. However, hybrids displayed a much greater metabolic activity, enhancing glycolysis to proliferate. Interestingly, modifying the metabolic environmental conditions through the use of 5-aminoimidazole-4-carbox-amide-1-ß-D-ribofuranoside (AICAR), an activator of the 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), specifically reduced the growth of hybrids, and also abrogated the invasive capacity of hybrids displaying enhanced glycolysis. Furthermore, AICAR efficiently blocked the tumoral features related to the aggressiveness of human UPS cell lines. CONCLUSION: Altogether, our findings strongly suggest that hybrids rely on higher energy flux to proliferate and that a drug altering this metabolic equilibrium could impair their survival and be potentially considered as a novel therapeutic strategy.

DRAG in situ barcoding reveals an increased number of HSPCs contributing to myelopoiesis with age.

Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we present an in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis rather than the increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remains constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity.

Distinct Cellular Origins and Differentiation Process Account for Distinct Oncogenic and Clinical Behaviors of Leiomyosarcomas.

In leiomyosarcoma (LMS), a very aggressive disease, a relatively transcriptionally uniform subgroup of well-differentiated tumors has been described and is associated with poor survival. The question raised how differentiation and tumor progression, two apparently antagonist processes, coexist and allow tumor malignancy. We first identified the most transcriptionally homogeneous LMS subgroup in three independent cohorts, which we named 'hLMS'. The integration of multi-omics data and functional analysis suggests that hLMS originate from vascular smooth muscle cells and show that hLMS transcriptional program reflects both modulations of smooth muscle contraction activity controlled by MYOCD/SRF regulatory network and activation of the cell cycle activity controlled by E2F/RB1 pathway. We propose that the phenotypic plasticity of vascular smooth muscle cells coupled with MYOCD/SRF pathway amplification, essential for hLMS survival, concomitant with PTEN absence and RB1 alteration, could explain how hLMS balance this uncommon interplay between differentiation and aggressiveness.

Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme.

Tumours are complex ecosystems composed of different types of cells that communicate and influence each other. While the critical role of stromal cells in affecting tumour growth is well established, the impact of mutant cancer cells on healthy surrounding tissues remains poorly defined. Here, using mouse intestinal organoids, we uncover a paracrine mechanism by which intestinal cancer cells reactivate foetal and regenerative YAP-associated transcriptional programmes in neighbouring wildtype epithelial cells, rendering them adapted to thrive in the tumour context. We identify the glycoprotein thrombospondin-1 (THBS1) as the essential factor that mediates non-cell-autonomous morphological and transcriptional responses. Importantly, Thbs1 is associated with bad prognosis in several human cancers. This study reveals the THBS1-YAP axis as the mechanistic link mediating paracrine interactions between epithelial cells in intestinal tumours.

Cell-cell fusion of mesenchymal cells with distinct differentiations triggers genomic and transcriptomic remodelling toward tumour aggressiveness.

Cell-cell fusion is a physiological process that is hijacked during oncogenesis and promotes tumour evolution. The main known impact of cell fusion is to promote the formation of metastatic hybrid cells following fusion between mobile leucocytes and proliferating tumour cells. We show here that cell fusion between immortalized myoblasts and transformed fibroblasts, through genomic instability and expression of a specific transcriptomic profile, leads to emergence of hybrid cells acquiring dissemination properties. This is associated with acquisition of clonogenic ability by fused cells. In addition, by inheriting parental properties, hybrid tumours were found to mimic the histological characteristics of a specific histotype of sarcomas: undifferentiated pleomorphic sarcomas with incomplete muscular differentiation. This finding suggests that cell fusion, as macroevolution event, favours specific sarcoma development according to the differentiation lineage of parent cells.

Tetraploidization of Immortalized Myoblasts Induced by Cell Fusion Drives Myogenic Sarcoma Development with DMD Deletion.

Whole-genome doubling is the second most frequent genomic event, after TP53 alterations, in advanced solid tumors and is associated with poor prognosis. Tetraploidization step will lead to aneuploidy and chromosomic rearrangements. The mechanism leading to tetraploid cells is important since endoreplication, abortive cytokinesis and cell fusion could have distinct consequences. Unlike processes based on duplication, cell fusion involves the merging of two different genomes, epigenomes and cellular states. Since it is involved in muscle differentiation, we hypothesized that it could play a role in the oncogenesis of myogenic cancers. Spontaneous hybrids, but not their non-fused immortalized myoblast counterparts they are generated from, induced tumors in mice. Unstable upon fusion, the hybrid genome evolved from initial mitosis to tumors with a highly rearranged genome. This genome remodeling finally produced targeted DMD deletions associated with replicative stress, isoform relocalization and metastatic spreading, exactly as observed in human myogenic sarcomas. In conclusion, these results draw a model of myogenic oncogenesis in which cell fusion and oncogene activation combine to produce pleomorphic aggressive sarcomas.

Genome remodeling upon mesenchymal tumor cell fusion contributes to tumor progression and metastatic spread.

Cell fusion in tumor progression mostly refers to the merging of a cancer cell with a cell that has migration and immune escape capabilities such as macrophages. Here we show that spontaneous hybrids made from the fusion of transformed mesenchymal cells with partners from the same lineage undergo nonrecurrent large-scale genomic rearrangements, leading to the creation of highly aneuploid cells with novel phenotypic traits, including metastatic spreading capabilities. Moreover, in contrast to their parents, hybrids were the only cells able to recapitulate in vivo all features of human pleomorphic sarcomas, a rare and genetically complex mesenchymal tumor. Hybrid tumors not only displayed specific mesenchymal markers, but also combined a complex genetic profile with a highly metastatic behavior, like their human counterparts. Finally, we provide evidence that patient-derived pleomorphic sarcoma cells are inclined to spontaneous cell fusion. The resulting hybrids also gain in aggressiveness, exhibiting superior growth capacity in mouse models. Altogether, these results indicate that cell fusion has the potential to promote cancer progression by increasing growth and/or metastatic capacities, regardless of the nature of the companion cell. Moreover, such events likely occur upon sarcoma development, paving the way for better understanding of the biology, and aggressiveness of these tumors.

Fusion-mediated chromosomal instability promotes aneuploidy patterns that resemble human tumors.

Oncogenesis is considered to result from chromosomal instability, in addition to oncogene and tumor-suppressor alterations. Intermediate to aneuploidy and chromosomal instability, genome doubling is a frequent event in tumor development but the mechanisms driving tetraploidization and its impact remain unexplored. Cell fusion, one of the pathways to tetraploidy, is a physiological process involved in mesenchymal cell differentiation. Besides simple genome doubling, cell fusion results in the merging of two different genomes that can be destabilized upon proliferation. By testing whether cell fusion is involved in mesenchymal oncogenesis, we provide evidence that it induces genomic instability and mediates tumor initiation. After a latency period, the tumor emerges with the cells most suited for its development. Furthermore, hybrid tumor genomes were stabilized after this selection process and were very close to those of human pleomorphic mesenchymal tumors. Thus genome restructuring triggered by cell fusion may account for the chromosomal instability involved in oncogenesis.

Calpain-6 controls the fate of sarcoma stem cells by promoting autophagy and preventing senescence.

Sarcomas are still unsolved therapeutic challenges. Cancer stem cells are believed to contribute to sarcoma development, but lack of specific markers prevents their characterization and targeting. Here, we show that calpain-6 expression is associated with cancer stem cell features. In mouse models of bone sarcoma, calpain-6-expressing cells have unique tumor-initiating and metastatic capacities. Calpain-6 levels are especially high in tumors that have been successfully propagated in mouse to establish patient-derived xenografts. We found that calpain-6 levels are increased by hypoxia in vitro and calpain-6 is detected within hypoxic areas in tumors. Furthermore, calpain-6 expression depends on the stem cell transcription network that involves Oct4, Nanog, and Sox2 and is activated by hypoxia. Calpain-6 knockdown blocks tumor development in mouse and induces depletion of the cancer stem cell population. Data from transcriptomic analyses reveal that calpain-6 expression in sarcomas inversely correlates with senescence markers. Calpain-6 knockdown suppresses hypoxia-dependent prevention of senescence entry and also promotion of autophagic flux. Together, our results demonstrate that calpain-6 identifies sarcoma cells with stem-like properties and is a mediator of hypoxia to prevent senescence, promote autophagy, and maintain the tumor-initiating cell population. These findings open what we believe is a novel therapeutic avenue for targeting sarcoma stem cells.