State of the Art and New Trends from the Second International StemNet Meeting.

The Second International StemNet (Federation of Stem Cell Research Associations) meeting took place on 18-20 October 2023 in Brescia (Italy), with the support of the University of Brescia and the Zooprophylactic Institute of Lombardy and Emilia Romagna. The program of the meeting was articulated in nine sections: (1) Biomedical Communication in Italy: Critical Aspects; (2) StemNet Next Generation Session; (3) Cell-Free Therapies; (4) Tips and Tricks of Research Valorisation; (5) Stem Cells and Cancer; (6) Stem Cells in Veterinary Applications; (7) Stem Cells in Clinical Applications; (8) Organoids and 3D Systems; (9) induced pluripotent stem cells (iPCS) and Gene Therapy. National and International speakers presented their scientific works, inspiring debates and discussions among the attendees. The participation in the meeting was high, especially because of the young researchers who animated all the sessions and the rich poster session.

Why do certain cancer cells alter functionality and fuse?

Cancer cell fusion represents a rare event. However, the surviving cancer hybrid cells after a post-hybrid selection process (PHSP) can overgrow other cancer cells by exhibiting a proliferation advantage and/or expression of cancer stem-like properties. Addition of new tumor properties during hetero-fusion of cancer cells e.g. with mesenchymal stroma-/stem-like cells (MSC) contribute to enhanced tumor plasticity via acquisition of new/altered functionalities. This provides new avenues for tumor development and metastatic behavior. Consequently, the present review article will also address the question as to whether cancer cell fusion represents a general and possibly evolutionary-conserved program or rather a random process?

Intrinsic signalling factors associated with cancer cell-cell fusion.

Cellular fusion e.g. between cancer cells and normal cells represents a stepwise process that is tightly regulated. During a pre-hybrid preparation program somatic cells and/or cancer cells are promoted to a pro-fusogenic state as a prerequisite to prepare a fusion process. A pro-fusogenic state requires significant changes including restructure of the cytoskeleton, e.g., by the formation of F-actin. Moreover, distinct plasma membrane lipids such as phosphatidylserine play an important role during cell fusion. In addition, the expression of distinct fusogenic factors such as syncytins and corresponding receptors are of fundamental importance to enable cellular mergers. Subsequent hybrid formation and fusion are followed by a post-hybrid selection process. Fusion among normal cells is important and often required during organismal development. Cancer cells fusion appears more rarely and is associated with the generation of new cancer hybrid cell populations. These cancer hybrid cells contribute to an elevated tumour plasticity by altered metastatic behaviour, changes in therapeutic and apoptotic responses, and even in the formation of cancer stem/ initiating cells. While many parts within this multi-step cascade are still poorly understood, this review article predominantly focusses on the intracellular necessities for fusion among cancer cells or with other cell populations of the tumour microenvironment. Video Abstract.

Extracellular Events Involved in Cancer Cell-Cell Fusion.

Fusion among different cell populations represents a rare process that is mediated by both intrinsic and extracellular events. Cellular hybrid formation is relayed by orchestrating tightly regulated signaling pathways that can involve both normal and neoplastic cells. Certain important cell merger processes are often required during distinct organismal and tissue development, including placenta and skeletal muscle. In a neoplastic environment, however, cancer cell fusion can generate new cancer hybrid cells. Following survival during a subsequent post-hybrid selection process (PHSP), the new cancer hybrid cells express different tumorigenic properties. These can include elevated proliferative capacity, increased metastatic potential, resistance to certain therapeutic compounds, and formation of cancer stem-like cells, all of which characterize significantly enhanced tumor plasticity. However, many parts within this multi-step cascade are still poorly understood. Aside from intrinsic factors, cell fusion is particularly affected by extracellular conditions, including an inflammatory microenvironment, viruses, pH and ionic stress, hypoxia, and exosome signaling. Accordingly, the present review article will primarily highlight the influence of extracellular events that contribute to cell fusion in normal and tumorigenic tissues.

Spontaneous Fusion of MSC with Breast Cancer Cells Can Generate Tumor Dormancy.

Direct cellular interactions of MDA-MB-231cherry breast cancer cells with GFP-transduced human mesenchymal stroma/stem-like cells (MSCGFP) in a co-culture model resulted in spontaneous cell fusion by the generation of MDA-MSC-hyb5cherry GFP breast cancer hybrid cells. The proliferative capacity of MDA-MSC-hyb5 cells was enhanced about 1.8-fold when compared to the parental MDA-MB-231cherry breast cancer cells. In contrast to a spontaneous MDA-MB-231cherry induced tumor development in vivo within 18.8 days, the MDA-MSC-hyb5 cells initially remained quiescent in a dormancy-like state. At distinct time points after injection, NODscid mice started to develop MDA-MSC-hyb5 cell-induced tumors up to about a half year later. Following tumor initiation, however, tumor growth and formation of metastases in various different organs occurred rapidly within about 10.5 days. Changes in gene expression levels were evaluated by RNA-microarray analysis and revealed certain increase in dormancy-associated transcripts in MDA-MSC-hyb5. Chemotherapeutic responsiveness of MDA-MSC-hyb5 cells was partially enhanced when compared to MDA-MB-231 cells. However, some resistance, e.g., for taxol was detectable in cancer hybrid cells. Moreover, drug response partially changed during the tumor development of MDA-MSC-hyb5 cells; this suggests the presence of unstable in vivo phenotypes of MDA-hyb5 cells with increased tumor heterogeneity.

Human MSC-Derived Exosomes Reduce Cellular Senescence in Renal Epithelial Cells.

Cellular senescence of renal tubular cells is associated with chronic diseases and age-related kidney disorders. Therapies to antagonize senescence are, therefore, explored as novel approaches in nephropathy. Exosomes derived from human mesenchymal stroma-/stem-like cells (MSC) entail the transfer of multiple bioactive molecules, exhibiting profound regenerative potential in various tissues, including therapeutic effects in kidney diseases. Here, we first demonstrate that exosomes promote proliferation and reduce senescence in aged MSC cultures. For potential therapeutic perspectives in organ rejuvenation, we used MSC-derived exosomes to antagonize senescence in murine kidney primary tubular epithelial cells (PTEC). Exosome treatment efficiently reduced senescence while diminishing the transcription of senescence markers and senescence-associated secretory phenotype (SASP) factors. Concomitantly, we observed less DNA damage foci and more proliferating cells. These data provide new information regarding the therapeutic property of MSC exosomes in the development of renal senescence, suggesting a contribution to a new chapter of regenerative vehicles in senotherapy.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

Anti-Tumor Effects of Exosomes Derived from Drug-Incubated Permanently Growing Human MSC.

Similar to growth-limited human primary cultures of mesenchymal stroma/stem-like cells (MSC), the continuously proliferating human MSC544 cell line produced extracellular vesicles as characterized by expression of the tetraspanin molecules CD9, CD63, and CD81. Release of these particles was predominantly detectable during continuous cell growth of MSC544 in contrast to confluency-mediated transient growth arrest. For therapeutic use, these particles were isolated from proliferating MSC544 after taxol treatment and applied to different cancer cell cultures. A pronounced cytotoxicity of lung, ovarian, and breast cancer cells was observed primarily with taxol-loaded exosomes, similar to the effects displayed by application of taxol substance. While these findings suggested pronounced cancer cell targeting of MSC544 exosomes, a tumor therapeutic approach was performed using a mouse in vivo breast cancer model. Thus, intravenous injection of taxol-loaded MSC544 exosomes displayed superior tumor-reducing capabilities as compared to application of taxol exosomes by oral gavage. To broaden this therapeutic spectrum, epirubicin was applied to MSC544, and the derived exosomes likewise exhibited significant cytotoxic effects in different cancer cell cultures. These findings suggest an unlimited source for large-scale exosome production with reproducible quality to enable variable drug targeting of tumors or other diseases.

Altered Tumor Plasticity after Different Cancer Cell Fusions with MSC.

While cell fusion demonstrates an important pathway during tissue development and regeneration of distinct organs, this process can also contribute to pathophysiological phenotypes during tumor progression. Hybrid cell formation after heterofusion between cancer cells and various other cell types within the tumor microenvironment is observed in vitro and in vivo. In particular, mesenchymal stroma/stem-like cells (MSC) perform diverse levels of communication with cancer cells by exhibiting anti- and pro-tumorigenic effects. During these cellular interactions, MSC can eventually fuse with cancer cells. Thereby, the newly generated disparate hybrid populations display aneuploidy associated with chromosomal instability. Based upon a subsequent post-hybrid selection process (PHSP), fused cancer cells can undergo apoptosis/necroptosis, senescence, dormancy, or a proliferative state by acquisition of new properties. Consequently, PHSP-surviving hybrid cancer cells demonstrate altered functionalities within the tumor tissue. This is accompanied by changes in therapeutic responsiveness and a different metastatic behavior. Accordingly, enhanced tumor plasticity interferes with successful therapeutic interventions and aggravates patient prognoses. The present review article focusses on fusion of MSC with different human cancer cells, in particular breast cancer populations and resulting characteristics of various cancer hybrid cells. Moreover, some mechanisms of cancer cell fusion are discussed together with multiple PHSP pathways.

Reversible Growth-Arrest of a Spontaneously-Derived Human MSC-Like Cell Line.

Life cycle limitation hampers the production of high amounts of primary human mesenchymal stroma-/stem-like cells (MSC) and limits cell source reproducibility for clinical applications. The characterization of permanently growing MSC544 revealed some differentiation capacity and the simultaneous presence of known MSC markers CD73, CD90, and CD105 even after continuous long-term culture for more than one year and 32 passages. The expression of CD13, CD29, CD44, and CD166 were identified as further surface proteins, all of which were also simultaneously detectable in various other types of primary MSC populations derived from the umbilical cord, bone marrow, and placenta suggesting MSC-like properties in the cell line. Proliferating steady state MSC544 exhibited immune-modulatory activity similar to a subpopulation of long-term growth-inhibited MSC544 after 189d of continuous culture in confluency. This confluent connective cell layer with fibroblast-like morphology can spontaneously contract and the generated space is subsequently occupied by new cells with regained proliferative capacity. Accordingly, the confluent and senescence-associated beta-galactosidase-positive MSC544 culture with about 95% G0/G1 growth-arrest resumed re-entry into the proliferative cell cycle within 3d after sub-confluent culture. The MSC544 cells remained viable during confluency and throughout this transition which was accompanied by marked changes in the release of proteins. Thus, expression of proliferation-associated genes was down-modulated in confluent MSC544 and re-expressed following sub-confluent conditions whilst telomerase (hTERT) transcripts remained detectable at similar levels in both, confluent growth-arrested and proliferating MSC544. Together with the capability of connective cell layer formation for potential therapeutic approaches, MSC544 provide a long term reproducible human cell source with constant properties.

In Vitro Fusion of Normal and Neoplastic Breast Epithelial Cells with Human Mesenchymal Stroma/Stem Cells Partially Involves Tumor Necrosis Factor Receptor Signaling.

Formation of hybrid cells by "accidental cell fusion" of normal and neoplastic breast epithelial cells with local tissue-associated mesenchymal stroma/stem-like cells (MSC) in an inflammatory microenvironment can generate new cancer cell populations whereby molecular signaling mechanisms of this process remain unclear. Fusions of lentiviral enhanced green fluorescent protein-labeled MSC with mcherry-labeled breast epithelial cells were quantified and effects of tumor necrosis factor alpha (TNF-α) and receptor downstream signaling were investigated. Cocultures of MSC with normal human mammary epithelial cells, with neoplastic MCF10A, or with MDA-MB-231 or MCF7 breast cancer cells demonstrated hybrid cell formation between 0.1% and about 2% of the populations within 72 hours, whereby the fusion process occurred in less than 5 minutes. Addition of the pro-inflammatory cytokine TNF-α significantly enhanced MCF10A-MSC cell fusion. Small-interfering RNA (siRNA) knockdown experiments revealed an involvement of tumor necrosis factor (TNF) receptor-1 and -2 in this process. This was also substantiated by siRNA knockdown of tumor necrosis factor receptor type 1-associated death domain which abolished TNF-α-stimulated fusion. While TNF receptor signaling can be relayed via the Mitogen-activated protein kinase 8 (MAPK8), NF-κB or cell death pathways, examination of further downstream signaling exhibited little if any effects of MAPK8 or RelA (p65) on TNF-α-mediated cell fusion, respectively. These data suggested that cell fusion between MSC and MCF10A breast epithelial cells can be stimulated by TNF-α involving TNF receptor-activated cell death pathways or additional NF-κB signaling. Stem Cells 2018;36:977-989.

Concise Review: Crosstalk of Mesenchymal Stroma/Stem-Like Cells with Cancer Cells Provides Therapeutic Potential.

Various direct and indirect cellular interactions between multi-functional mesenchymal stroma/stem-like cells (MSCs) and cancer cells contribute to increasing plasticity within the tumor tissue and its microenvironment. Direct and tight communication between MSC and cancer cells is based on membrane protein interactions and the exchange of large plasma membrane fragments also known as trogocytosis. An ultimate but rare direct interaction resumes in fusion of these two cellular partners resulting in the formation of new cancer hybrid cell populations. Alternatively, indirect interactions are displayed by the release of membranous vesicle-encapsulated microRNAs and proteins or soluble components such as molecular growth factors, hormones, chemo-/cytokines, and metabolites. Released single molecules as well as multivesicular bodies including exosomes and microvesicles can form local concentration gradients within the tumor microenvironment and are incorporated not only by adjacent neighboring cells but also affect distant target cells. The present Review will focus on vesicle-mediated indirect communication and on cancer cell fusion with direct contact between MSC and cancer cells. These different types of interaction are accompanied by functional interference and mutual acquisition of new cellular properties. Consequently, alterations in cancer cell functionalities paralleled by the capability to reorganize the tumor stroma can trigger changes in metastatic behavior and promote retrodifferentiation to develop new cancer stem-like cells. However, exosomes and microvesicles acting over long distances may also provide a tool with therapeutic potential when loaded with anti-tumor cargo. Stem Cells 2018;36:951-968.

Involvement of Actin Cytoskeletal Components in Breast Cancer Cell Fusion with Human Mesenchymal Stroma/Stem-Like Cells.

Cell fusion as a rare event was observed following the co-culture of human MDA-MB-231cherry breast cancer cells or benign neoplastic MCF10Acherry breast epithelial cells together with different mesenchymal stroma/stem-like cells (MSCGFP) cultures, respectively, resulting in the generation of double-fluorescing hybrid cells. Analysis of potential molecular mechanisms for the formation of cancer hybrid cells revealed cytoskeletal components, including F-actin. Thus, a sub-lethal concentration of cytochalasin D, which blocks elongation of actin filaments, was able to significantly reduce cancer hybrid cell formation. Simultaneously, cell cycle progression of the different co-cultures remained unaffected following treatment with cytochalasin D, indicating continued proliferation. Moreover, exposure to 50 nM cytochalasin D revealed little if any effect on the expression of various integrins and cell adhesion molecules in the different co-cultures. However, LC-MS proteome analysis of the different control co-cultures compared to corresponding cytochalasin-treated co-cultures demonstrated predominant differences in the expression of actin-associated cytoskeletal proteins. In addition, the requirement of structured actin to provide an appropriate cytoskeletal network for enabling subsequent fusion processes was also substantiated by the actin filament disrupting latrunculin B, which inhibits the fusion process between the breast cancer populations and mesenchymal stroma/stem-like cells (MSC). Together, these findings suggest an important role of distinct actin structures and associated cytoskeletal components during cell fusion and the formation of breast cancer hybrid cells.

Changes in uPA, PAI-1, and TGF-ß Production during Breast Cancer Cell Interaction with Human Mesenchymal Stroma/Stem-Like Cells (MSC).

The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231cherry breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544GFP stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures. Moreover, uPA levels in 3D tumor spheroids remained elevated 9.4-fold above the average of five different human MSC cultures. In contrast, the expression of the corresponding plasminogen activator inhibitor type-1 (PAI-1) declined by 2.6-fold in the breast cancer cells and was even further reduced by 3.2-fold in the MDA-MB-231cherry/MSC544GFP 3D co-culture spheroids when compared to the various MSC populations. The supportive data were obtained for the production of TGF-ß1, which is an important growth factor in the regulation of tumor growth and metastasis formation. Whereas, TGF-ß1 release in MDA-MB-231cherry/MSC544GFP co-cultures was elevated by 1.56-fold as compared to MSC544 mono-cultures after 24 h; this ratio further increased to 2.19-fold after 72 h. Quantitative PCR analyses in MSC544 and MDA-MB-231 cells revealed that MSC, rather than the breast cancer cells, are responsible for TGF-ß1 synthesis and that TGF-ß1 contributes to its own synthesis in these cells. These findings suggested potential synergistic effects in the expression/secretion of uPA, PAI-1, and TGF-ß during the co-culture of breast cancer cells with MSC.

MSC stimulate ovarian tumor growth during intercellular communication but reduce tumorigenicity after fusion with ovarian cancer cells.

The tumor microenvironment enables important cellular interactions between cancer cells and recruited adjacent populations including mesenchymal stroma/stem cells (MSC). In vivo cellular interactions of primary human MSC in co-culture with human SK-OV-3 ovarian cancer cells revealed an increased tumor growth as compared to mono-cultures of the ovarian cancer cells. Moreover, the presence of MSC stimulated formation of liver metastases. Further interactions of MSC with the ovarian cancer cells resulted in the formation of hybrid cells by cell fusion. Isolation and single cell cloning of these hybrid cells revealed two differentially fused ovarian cancer cell populations termed SK-hyb1 and SK-hyb2. RNA microarray analysis demonstrated expression profiles from both parental partners whereby SK-hyb1 were attributed with more SK-OV-3 like properties and SK-hyb2 cells displayed more similarities to MSC. Both ovarian cancer hybrid populations exhibited reduced proliferative capacity compared to the parental SK-OV-3 cells. Moreover, the fused populations failed to develop tumors in NODscid mice. Together, these data suggested certain stimulatory effects on ovarian tumor growth in the presence of MSC. Conversely, fusion of MSC with SK-OV-3 cells contributed to the generation of new cancer hybrid populations displaying a significantly reduced tumorigenicity.

Enhanced metastatic capacity of breast cancer cells after interaction and hybrid formation with mesenchymal stroma/stem cells (MSC).

BACKGROUND: Fusion of breast cancer cells with tumor-associated populations of the microenvironment including mesenchymal stroma/stem-like cells (MSC) represents a rare event in cell communication whereby the metastatic capacity of those hybrid cells remains unclear. METHODS: Functional changes were investigated in vitro and in vivo following spontaneous fusion and hybrid cell formation between primary human MSC and human MDA-MB-231 breast cancer cells. Thus, lentiviral eGFP-labeled MSC and breast cancer cells labeled with mcherry resulted in dual-fluorescing hybrid cells after co-culture. RESULTS: Double FACS sorting and single cell cloning revealed two different aneuploid male hybrid populations (MDA-hyb1 and MDA-hyb2) with different STR profiles, pronounced telomerase activities, and enhanced proliferative capacities as compared to the parental cells. Microarray-based mRNA profiling demonstrated marked regulation of genes involved in epithelial-mesenchymal transition and increased expression of metastasis-associated genes including S100A4. In vivo studies following subcutaneous injection of the breast cancer and the two hybrid populations substantiated the in vitro findings by a significantly elevated tumor growth of the hybrid cells. Moreover, both hybrid populations developed various distant organ metastases in a much shorter period of time than the parental breast cancer cells. CONCLUSION: Together, these data demonstrate spontaneous development of new tumor cell populations exhibiting different parental properties after close interaction and subsequent fusion of MSC with breast cancer cells. This formation of tumor hybrids contributes to continuously increasing tumor heterogeneity and elevated metastatic capacities.

Cancer stem cell niche models and contribution by mesenchymal stroma/stem cells.

BACKGROUND: The initiation and progression of malignant tumors is driven by distinct subsets of tumor-initiating or cancer stem-like cells (CSCs) which develop therapy/apoptosis resistance and self-renewal capacity. In order to be able to eradicate these CSCs with novel classes of anti-cancer therapeutics, a better understanding of their biology and clinically-relevant traits is mandatory. MAIN BODY: Several requirements and functions of a CSC niche physiology are combined with current concepts for CSC generation such as development in a hierarchical tumor model, by stochastic processes, or via a retrodifferentiation program. Moreover, progressive adaptation of endothelial cells and recruited immune and stromal cells to the tumor site substantially contribute to generate a tumor growth-permissive environment resembling a CSC niche. Particular emphasis is put on the pivotal role of multipotent mesenchymal stroma/stem cells (MSCs) in supporting CSC development by various kinds of interaction and cell fusion to form hybrid tumor cells. CONCLUSION: A better knowledge of CSC niche physiology may increase the chances that cancer stemness-depleting interventions ultimately result in arrest of tumor growth and metastasis.

c-Met expression and activity in urogenital cancers - novel aspects of signal transduction and medical implications.

C-Met is a receptor tyrosine kinase with multiple functions throughout embryonic development, organogenesis and wound healing and is expressed in various epithelia. The ligand of c-Met is Hepatocyte Growth Factor (HGF) which is secreted among others by mesenchymal stroma/stem (MSC) cells.Physiological c-Met functions are centred around processes that underly cellular motility and invasive growth. Aberrant c-Met expression and activity is observed in numerous cancers and makes major contributions to cell malignancy. Importantly, HGF/c-Met signaling is crucial in the context of communication between cancer cells and the the tumor stroma.Here, we review recent findings on roles of dysregulated c-Met in urogenital tumors such as cancers of the urinary bladder, prostate, and ovary. We put emphasis on novel aspects of cancer-associated c-Met expression regulation on both, HGF-dependent and HGF-independent non-canonical mechanisms. Moreover, this review focusses on c-Met-triggered signalling with potential relevance for urogenital oncogenesis, and on strategies to specifically inhibit c-Met activity.

The role of TGF-ß and its crosstalk with RAC1/RAC1b signaling in breast and pancreas carcinoma.

This article focusses on the role of TGF-ß and its signaling crosstalk with the RHO family GTPases RAC1 and RAC1b in the progression of breast and pancreatic carcinoma. The aggressive nature of these tumor types is mainly due to metastatic dissemination. Metastasis is facilitated by desmoplasia, a peculiar tumor microenvironment and the ability of the tumor cells to undergo epithelial-mesenchymal transition (EMT) and to adopt a motile and invasive phenotype. These processes are controlled entirely or in part by TGF-ß and the small RHO GTPase RAC1 with both proteins acting as tumor promoters in late-stage cancers. Data from our and other studies point to signaling crosstalk between TGF-ß and RAC1 and the related isoform, RAC1b, in pancreatic and mammary carcinoma cells. Based on the exciting observation that RAC1b functions as an endogenous inhibitor of RAC1, we propose a model on how the relative abundance or activity of RAC1 and RAC1b in the tumor cells may determine their responses to TGF-ß and, ultimately, the metastatic capacity of the tumor.

TGF-ß-Dependent Growth Arrest and Cell Migration in Benign and Malignant Breast Epithelial Cells Are Antagonistically Controlled by Rac1 and Rac1b.

Despite improvements in diagnosis and treatment, breast cancer is still the most common cancer type among non-smoking females. TGF-ß can inhibit breast cancer development by inducing cell cycle arrest in both, cancer cells and, as part of a senescence program in normal human mammary epithelial cells (HMEC). Moreover, TGF-ß also drives cell migration and invasion, in part through the small GTPases Rac1 and Rac1b. Depletion of Rac1b or Rac1 and Rac1b in MDA-MB-231 or MDA-MB-435s breast cancer cells by RNA interference enhanced or suppressed, respectively, TGF-ß1-induced migration/invasion. Rac1b depletion in MDA-MB-231 cells also increased TGF-ß-induced p21WAF1 expression and ERK1/2 phosphorylation. Senescent HMEC (P15/P16), when compared to their non-senescent counterparts (P11/P12), presented with dramatically increased migratory activity. These effects were paralleled by elevated expression of genes associated with TGF-ß signaling and metastasis, downregulated Rac1b, and upregulated Rac1. Our data suggest that acquisition of a motile phenotype in HMEC resulted from enhanced autocrine TGF-ß signaling, invasion/metastasis-associated gene expression, and a shift in the ratio of antimigratory Rac1b to promigratory Rac1. We conclude that although enhanced TGF-ß signaling is considered antioncogenic in HMEC by suppressing oncogene-induced transformation, this occurs at the expense of a higher migration and invasion potential.